田克恭-PCV3、PEDV基因工程亞單位疫苗的創制.pdf

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1、PCV3、PEDV基基因因工工程程亞亞單單位位疫疫苗苗的的創創制制Development of Genetic Engineering Subunit Vaccine of PCV3 and PEDV普萊柯生物工程股份有限公司國家獸用藥品工程技術研究中心Pulike Biological Engineering Co.,Ltd.National Engineering Research Center for Veterinary Drugs田田克克恭恭Tian Kegong2024.04.26 鄭州April 26,2024 Zhengzhou一帶一路國際生豬產業大會IPCV3亞單位疫苗的創制

2、Development of PCV3 Subunit Vaccine二IIPEDV亞單位疫苗的創制Development of PEDV Subunit Vaccine目錄Table of Contents一帶一路國際生豬產業大會PCV3亞亞單單位位疫疫苗苗的的創創制制Development of PCV3 Subunit VaccineI一帶一路國際生豬產業大會主主要要內內容容Main contents2.PCV3 VLPs的的表表達達2.Expression of PCV3 VLPs3.PCV3 VLPs工工藝藝開開發發3.PCV3 VLPs Process Development1.P

3、CV3概概述述1.Overview of PCV3PCV3病病原原學學分分析析PCV3 Etiological AnalysisPCV的的發發現現史史History of PCVPCV3流流行行現現狀狀Epidemiological status of PCV3Cap序序列列分分析析、優優化化Cap sequence analysis and optimization表表達達載載體體改改造造Expression vector modification表表達達細細胞胞構構建建Expression Cell ConstructionPCV3 VLPs表表征征PCV3 VLPs Characteri

4、zationPCV3 VLPs表表達達工工藝藝開開發發PCV3 VLPs Expression Process DevelopmentPCV3 VLPs純純化化工工藝藝開開發發PCV3 VLPs Purification Process Development5.PCV3 VLPs疫疫苗苗創創制制5.PCV3 VLPs vaccine development疫疫苗苗安安全全性性Vaccine safety疫疫苗苗效效力力Vaccine efficacy4.PCV3診診斷斷試試劑劑開開發發4.Development of PCV3 diagnostic reagent特特異異性性單單抗抗制制備備

5、Preparation of specific monoclonal antibodies阻阻斷斷ELISA的的開開發發Development of Blocking ELISA阻阻斷斷ELISA的的評評價價Evaluation of Blocking ELISA阻阻斷斷ELISA的的臨臨床床應應用用Clinical application of blocking ELISA一帶一路國際生豬產業大會病病原原：PCV3屬于圓環病毒科、圓環病毒屬成員；通過呼呼吸吸道道和消消化化道道傳播。Pathogen:PCV3 belongs to the Circoviridae family and the

6、 Circovirus genus;spread through respiratory tractand Digestive tract.臨臨床床表表現現和和危危害害：母豬體表多處出現丘疹性皮炎，可造成懷孕母豬流產、死胎和木乃伊胎等；斷斷奶奶仔仔豬豬表現為食食欲欲下下降降、發發熱熱和和先先天天性性震震顫顫等；所所有有年年齡齡段段仔豬均可感染，小鼠、犬、牛亦可感染。Clinical manifestations and hazards:papular dermatitis appears on many parts of the sows body surface,which can caus

7、e miscarriage,stillbirth and mummification of pregnant sows;Weaned pigletsManifested as Loss of appetite,fever,and congenital tremor,etc;All Ages of Piglets can be infected,as can mice,dogs and cattle.病病理理損損傷傷：主要表現在皮膚真皮和皮下組織出現壞壞死死性性血血管管炎炎，呈纖維蛋白樣變，淋巴結出現肉肉芽芽腫腫性性淋淋巴巴結結炎炎。Pathological damage:mainly mani

8、fested as necrotizing vasculitis in the skin,dermis,and subcutaneous tissue,presenting as fibroid like changes,and granulomatous lymphadenitis in lymph nodesPCV3基因組結構PCV3 Genome StructurePalinski R,et al JVI 2016.1、PCV3概概述述1.Overview of PCV3一帶一路國際生豬產業大會1、PCV3概概述述1.Overview of PCV315.17%13.21%33.33%2

9、11666962022年年2023年年2024年年3月月Mar.2024樣樣品品檢檢測測量量Amount of sample tested陽陽性性率率Positive ratePCV3流流行行情情況況PCV3 prevalence國家中心2022-2024年監測數據，以及其他11家單位的監測數據顯示：豬圓環病毒3型臨床檢測陽性率逐年上升。The monitoring data of the National Center from 2022 to 2024,as well as the monitoring data of 11 other units,show:Porcine circovi

10、rus type 3 positive rate of clinical detection has increased year by year.海南Hainan黑龍江Heilongjiang吉林Jilin遼寧Liaoning山東Shandong福建Fujian江西Jiangxi安徽Anhui湖北Hubei湖南Hunan廣東Guangdong廣西Guangxi上海Shanghai河南Henan山西Shanxi內蒙古Inner Mongolia陜西Shaanxi寧夏Ningxia甘肅Gansu四川Sichuan貴州Guizhou云南Yunnan西藏Tibet新疆Xinjiang江蘇Jiangs

11、u浙江Zhejiang北京Beijing天津Tianjin河北Hebei青海Qinghai18.13%15.89%25.10%23.20%23.20%8.81%9.75%2.17%33.45%24.17%26.30%PCV3臨臨床床陽陽性性率率Clinical positive rate of PCV3數據來源：公眾號和參考文獻Data source:Official account and references數據來源：國家獸用藥品工程技術研究中心Data source:National Engineering and Technology Research Center for Veter

12、inary Drugs一帶一路國際生豬產業大會2017197419982019PCV1PCV2PCV3PCV4首個PCV2疫苗The first PCV2 vaccine普普萊萊柯柯PCV2全全病病毒毒滅滅活活苗苗Pulike PCV2 whole virus inactivated vaccine普普萊萊柯柯PCV2亞亞單單位位疫疫苗苗Pulike PCV2 subunit vaccine對豬無致病性Not pathogenic to swineBrian M,et al JGV，1998.Palinski R,et al JVI 2016.PDNS的母豬和流產胎兒PDNS sows and

13、 aborted fetuses檢測到PCV3核酸PCV3 nucleic acid detected斷奶仔豬多系統衰竭綜合征（PMWS）,皮炎與腎病綜合征(PNDS)的主要病原main pathogen of postweaning multisystemic wasting syndrome(PMWS),dermatitis and nephrotic syndrome(PNDS）對豬致病性待進一步研究Pathogenicity to swine requires further study200920102016截至目前共約30+家Up to now about 30+PCV2疫苗上市P

14、CV2 vaccine launched1、PCV3概概述述1.Overview of PCV3PCV2疫苗的廣泛使用，有效控制了臨床上PCV2造成的危害。The widespread use of PCV2 vaccine has effectively controlled the harm caused by PCV2 in clinical practice.PCV3發現至今接近10年，流行率逐年升高，亟需有效的疫苗進行防控。It has been nearly 10 years since the discovery of PCV3,and the prevalence is inc

15、reasing year by year.Therefore,effective vaccines are urgently needed for prevention and control.一帶一路國際生豬產業大會2、PCV3 VLPs的的表表達達2.Expression of PCV3 VLPsNLS1NLS2NLS3NLS4NLS5NLS6注：數據未公開Note:Data are not publicly available疏水性分析Hydrophobicity analysis基于BepiPred-2.0的抗原決定簇預測Antigenic determinant prediction

16、 based on BepiPred-2.0ORF2編碼的Cap為唯一的結構蛋白，Cap蛋白無強疏水性片段，抗原決定簇均勻分布。Cap encoded by ORF2 is a unique structural protein.The Cap protein has no strongly hydrophobic fragments,and the antigenic determinants are evenly distributed.2.1 Cap序序列列分分析析、優優化化2.1 Cap sequence analysis and optimization通過對Cap蛋白不不同同結結構

17、構功功能能域域的的解解析析和多多種種表表達達策策略略的的驗驗證證，確定VLPs表表達達效效率率最最高高的表達策略。By analyzing the different structural and functional domains of Cap protein and validating multiple expression strategies,the expression strategy with the highest efficiency of VLPs was determined.一帶一路國際生豬產業大會基于Phyre2的二級結構預測Secondary Structure

18、 Prediction Based on Phyre2基于PCV3 Cap蛋蛋白白序序列列和和結結構構分分析析，PCV3 Cap的CD loop密度較低，結構構象與PCV2衣殼蛋白明顯不同，優選昆昆蟲蟲細細胞胞-桿桿狀狀病病毒毒表表達達系統進行 Cap蛋白的表達。Based on PCV3 Cap Protein sequence and structure analysisThe CD loop density of PCV3 Cap is low,and its structural conformation is obviously different from that of PCV2

19、 capsid protein.Insect cell-baculovirus expression was used to express Cap protein.基于Chou&Fasman的二級結構預測Secondary Structure Prediction Based on Chou&FasmanPCV2和 PCV3 Cap三級結構對比 Bi Mingfang,et al.Bioscience Reports,2020,40(6):BSR20201109Comparison of the tertiary structures of PCV2 and PCV3 Cap Bi Ming

20、fang,et al.Bioscience Reports,2020,40(6):BSR202011092、PCV3 VLPs的的表表達達2.Expression of PCV3 VLPs2.1 Cap序序列列分分析析、優優化化2.1 Cap sequence analysis and optimization一帶一路國際生豬產業大會針針對對昆昆蟲蟲細細胞胞-桿桿狀狀病病毒毒表表達達系統特性,優化PCV3 Cap基因的GC含含量量、密密碼碼子子使使用用頻頻率率和和RNA酶酶的的剪剪接接位位點點、RNA穩穩定定反反式式作作用用元元件件等影響基因表達的關鍵因素，提高蛋白表達量。The key fa

21、ctors affecting gene expression,such as GC content,codon frequency,splice site of RNAase and RNA stabilizing trans-acting element of PCV3 Cap gene,were optimized for the characteristics of insect cell-barcovirus expression system to improve the protein expression.目的基因結構與理化特性分析Analysis of target gene

22、 structure and physicochemical properties密碼子偏嗜性與RNA二級結構穩定性Codon bias and RNA secondary structure stability2、PCV3 VLPs的的表表達達2.Expression of PCV3 VLPs2.1 Cap序序列列分分析析、優優化化2.1 Cap sequence analysis and optimization一帶一路國際生豬產業大會同時敲除桿狀病毒中競競爭爭表表達達基因P10、阻阻礙礙蛋蛋白白分分泌泌的基因ChiA與v-cath以及影影響響遺遺傳傳穩穩定定性性的FP25K與DA26基因

23、轉座子整合位點。The competitively expressed gene P10,the genes ChiA and v-cath that block protein secretion,and the transposon integration sites of the FP25K and DA26 genes that affect genetic stability were also knocked down in baculoviruses.構建多多基基因因改改造造的穩定高效載體桿狀病毒，基因轉錄與蛋白分泌效率提升4倍以上，進進一一步步提提報報Cap蛋蛋白白的的表表達達

24、效效率率。Construction of a stable and efficient vector baculovirus with multi-gene modification,which enhances the efficiency of gene transcription and protein secretion by more than 4-fold,and further improves the expression efficiency of Cap proteins.ZL201710633480.92.2 表表達達載載體體改改造造2.2 Expression vect

25、or modification王同燕等.中國動物傳染病學報Wang Tongyan et al.Chinese Journal of Animal Infectious Diseases2、PCV3 VLPs的的表表達達2.Expression of PCV3 VLPs一帶一路國際生豬產業大會Sf21Sf9重組蛋白表達Expression of recombinant proteinHi5重組毒株的構建Construction of recombinant strains利用多多基基因因定定向向改改造造的昆蟲細胞-桿狀病毒表達系統，通過多重的篩選，實現PCV3 Cap蛋白的可溶表達。Multi

26、-gene targeted modified insect cell-baculovirus expression system was used to achieve soluble expression of PCV3 Cap protein through multiple screening.2、PCV3 VLPs的的表表達達2.Expression of PCV3 VLPsCap geneIFA identificationM Sf21 Sf9 Hi52.3 重重組組桿桿狀狀病病毒毒的的構構建建2.3 Construction of recombinant baculovirus一

27、帶一路國際生豬產業大會透射電鏡(TEM)觀察VLPs形態Morphology of VLPs observed by transmission electron microscope(TEM)動態光散射(DLS)分析VLPs顆粒粒徑、分散性Dynamic light scattering(DLS)analysis of particle size and dispersion of VLPs高效分子排阻色譜(HPSEC)檢測VLPs組裝效率Detection of VLPs Assembly Efficiency by High Performance Size Exclusion Chrom

28、atography(HPSEC)VLPsZ-Average:23.29nm PDI:0.105TEM、DLS檢測顯示Cap蛋白能高效自組裝為直徑約約20nm、均均一一、完完整整的的VLPs，HPSEC檢測VLPs組組裝裝效效率率接接近近100%。TEM and DLS assays showed that Cap proteins could self-assemble efficiently into homogeneous and intact VLPs with a diameter of about 20 nm,and the assembly efficiency of VLPs w

29、as close to 100%in HPSEC assay.2、PCV3 VLPs的的表表達達2.Expression of PCV3 VLPs2.4 PCV3 VLPs表表征征2.4 PCV3 VLPs Characterization一帶一路國際生豬產業大會獲獲中中國國專專利利6件件，美美國國專專利利2件件，日日本本專專利利4件件Obtained 6 Chinese patents,2 US patents and 4 Japanese patents完成PCV3 VLPs的冷冷凍凍電電鏡鏡結結構構解解析析，進進一一步步證證實實VLPs組組裝裝正正確確。Completion Cryo-e

30、lectron microscopy structure analysis of PCV3 VLPs,further confirmed that the VLPs were assembled correctly.ZL201710198414.3（中國）ZL201710433755.4（中國）ZL201710465252.5（中國）ZL201710464584.1（中國）ZL201710433747.X（中國）ZL201710294735.3（中國）US10869919B2（美國）US11116836B2（美國）JP6821818B2（日本）JP6914370B2（日本）JP7005747B

31、2（日本）JP6882602B2（日本）Deng Junhua,et al.Archives of Virology,2018,163(2):479-482 Bi Mingfang,et al.Bioscience Reports,2020,40(6):BSR20201109Deng Junhua,et al.Archives of Virology,2018,163(2):479-482 Bi Mingfang,et al.Bioscience Reports,2020,40(6):BSR20201109PCV3 VLPs cryo-EM圖像Cryo-EM images of PCV3 VL

32、Ps2、PCV3 VLPs的的表表達達2.Expression of PCV3 VLPs2.4 PCV3 VLPs表表征征2.4 PCV3 VLPs Characterization一帶一路國際生豬產業大會創建定定向向調調節節通通氣氣模模式式與半半連連續續滲滲透透壓壓動動態態平平衡衡補補料料工藝，精確調控培養過程營養代謝平衡，提升昆昆蟲蟲細細胞胞-桿桿狀狀病病毒毒系系統統表達重組蛋白的能力。The creation of directional regulation of ventilation mode and semi-continuous osmotic pressure dynamic

33、 balance replenishment process,precise regulation of nutritional and metabolic balance in the culture process,and enhancement of the ability of insect cell-barcovirus system to express recombinant proteins.ZL201010513446.6ZL201710633480.93.1 PCV3 VLPs表表達達工工藝藝開開發發3.1 PCV3 VLPs Expression Process Deve

34、lopment3、PCV3 VLPs工工藝藝開開發發3.Process development of PCV3 VLPs一帶一路國際生豬產業大會3、PCV3 VLPs工工藝藝開開發發3.Process development of PCV3 VLPsBIOSTAT RM反應器種子制備BIOSTAT RM Reactor Seed PreparationBIOSTAT D-DCU 150L工藝開發BIOSTAT D-DCU 150L Process Development20-500L細胞種子罐逐級放大Gradually scale up 20-500 L cell seed tank2000L

35、培養工藝驗證Validation of 2000 L culture process目前已在2000L規模實現PCV3 VLPs的全全自自動動、高高密密度度、高高可可溶溶性性表達。Fully automated,high-density,and highly soluble expression of PCV3 VLPs has been achieved at the 2000L scale.連續3批表達結果Expression results of 3 consecutive batches3.1 PCV3 VLPs表表達達工工藝藝開開發發3.1 PCV3 VLPs Expression

36、Process Development通過冷冷模模實實驗驗對反應器內部流流體體混混合合、流流體體剪剪切切和氣氣液液傳傳質質特性進行考察，開發了PCV3 VLPs培養工藝。The PCV3 VLPs incubation process was developed by examining the fluid mixing,fluid shear and gas-liquid mass transfer characteristics inside the reactor through cold modeling experiments.一帶一路國際生豬產業大會通過切切向向流流超超濾濾濃濃縮縮

37、工工藝藝和和層層析析工工藝藝優優化化，開發了PCV3 VLPs的高純度分離純化工藝，可規規模?；浦苽鋫涓吒呒兗兌榷萈CV3 VLPs。Through the optimization of tangential flow ultrafiltration concentration process and chromatography process,a high-purity separation and purification process for PCV3 VLPs was developed,which can be scaled up to prepare high-purit

38、y PCV3 VLPs.層層析析純純化化Chromatographic purification3.2 PCV3 VLPs純純化化工工藝藝開開發發3.2 PCV3 VLPs Purification Process Development 細細胞胞裂裂解解澄澄清清切切向向流流超超濾濾 Cell lysis and clarification Tangential flow ultrafiltration25kD12341：Marker 2：純化前1:Marker 2:Before purification3：粗純3:Crude purification4：精純4:Refining3、PCV3

39、VLPs工工藝藝開開發發3.Process development of PCV3 VLPs一帶一路國際生豬產業大會CD-loop(72-79aa)和EF-loop(109-131aa)暴露在PCV3 VLPs表面CD-loop(72-79aa)and EF-loop(109-131aa)were exposed on the surface of PCV3 VLPsmAb-1H11 識別VLP表面的CD-loop(72-79aa)MAb-1H11 recognizes CD-loop(72-79aa)on the surface of VLPsLi Xiangdong,et al.Journ

40、al of Virological Methods,2018,261:10-13 Bi Mingfang,et al.Bioscience Reports,2020,40(6):BSR20201109PCV3 VLPs的免疫電鏡 Immunoelectron microscopy of PCV3 VLPsChia-Chun Chang,et al.AMB Express,2023,13(1):141120-134aa位于病毒粒子表面120-134aa is located on the surface of virus particlesPCV3 Cap的3D模擬結構 3D simulatio

41、n structure of PCV3 CapJiang Min,et al.Applied Microbiology and Biotechnology,2020,104(14):6223-623457-61aa、140-146aa、161-166aa為線性B細胞表位57-61aa,140-146aa and 161-166aa are linear B cell epitopes140-146aa在PCVs中高度保守140-146aa is highly conserved in PCVs4.1 特特異異性性單單抗抗制制備備4.1 Preparation of specific monoc

42、lonal antibodies4、PCV3診診斷斷試試劑劑的的開開發發4.Development of PCV3 diagnostic reagents基于PCV3Cap抗抗原原表表位位分分析析和VLPs的的結結構構解解析析，篩選出暴露于VLPs表面的優優勢勢抗抗原原表表位位，并通過系統性親和篩選，定向篩選到識別優勢抗原表位CD-loop(72-79aa)的的特特異異性性單單抗抗。Based on PCV3 Cap antigenic epitope analysis and structural elucidation of VLPs,the predominant antigenic e

43、pitopes exposed on the surface of VLPs were screened,and specific monoclonal antibodies recognizing the predominant antigenic epitope,CD-loop(72-79aa),were targeted by systematic affinity screening.一帶一路國際生豬產業大會4.2 阻阻斷斷ELISA的的開開發發4.2 Development of Blocking ELISAPCV3 VLPs抗原包被板PCV3 VLPs antigen coated

44、 platePCV3 VLPs抗原包被板PCV3 VLPs antigen coated plate基于制備的PCV3特異性單單抗抗，開發了PCV3抗抗體體液液相相阻阻斷斷ELISA試試劑劑盒盒，N/P值值10。Based on the prepared PCV3-specific monoclonal antibody,a PCV3 antibody liquid phase blocking ELISA kit was developed with an N/P value of 10.PCV3 VLPs抗原包被板PCV3 VLPs antigen coated platePCV3 VLP

45、s抗原包被板PCV3 VLPs antigen coated plate底物溶液Substrate solution底物溶液Substrate solution不不顯顯色色或或顯顯色色淺淺No color or light color顯顯色色深深Deep color rendering酶酶標標單單抗抗Enzyme-labeled monoclonal antibody4、PCV3診診斷斷試試劑劑的的開開發發4.Development of PCV3 diagnostic reagentsPCV3抗體液相阻斷ELISA試劑盒PCV3 Antibody Liquid Phase Blocking

46、ELISA Kit一帶一路國際生豬產業大會陽陽性性樣樣本本Positive sample陰陰性性樣樣本本Negative sample050100阻阻斷斷率率（%）Blocking rate(%)120敏敏感感性性與與特特異異性性Sensitivity and specificity0.51.01.52.005101520重重復復性性Repeatability檢檢測測值值Measured valueCV值值（%）CV value(%)0 個個月月0 months3 個個月月3 months9 個個月月9 months1 2 個個月月1 2 months0201006 個個月月6 months保

47、保存存時間retention time5040實實時時穩穩定定性性Real-time stability阻阻 斷斷 率率（%）B l o c k i n g r a t e (%)70809060敏敏感感性性和和特特異異性性良良好好Good sensitivity and specificity重重復復性性好好，CV值值均均小小于于10%The repeatability was good,and the CV value was less than 10%.實實時時保保存存12個個月月，穩穩定定性性良良好好Real-time storage for 12 months,with good s

48、tability4.3 阻阻斷斷ELISA的的評評價價4.3 Evaluation of Blocking ELISA4、PCV3診診斷斷試試劑劑的的開開發發4.Development of PCV3 diagnostic reagents一帶一路國際生豬產業大會4.4 阻阻斷斷ELISA的的臨臨床床應應用用4.4 Clinical application of blocking ELISA檢測PCV3核酸陽性場血清，抗體陽性率較高Detect PCV3 nucleic acid positive sera,and the positive rate of antibody is high檢測

49、PCV3核酸陰性場血清，抗體結果均為陰性The PCV3 nucleic acid negative serum was tested,and the antibody results were all negativePCV3免疫場，二免后2周，抗體水平整齊，離散度較低PCV3 immune field,2 weeks after the second immunization,the antibody level is regular and the dispersion is low4、PCV3診診斷斷試試劑劑的的開開發發4.Development of PCV3 diagnostic

50、reagents一帶一路國際生豬產業大會5、PCV3 VLPs疫疫苗苗的的創創制制5.Development of PCV3 VLPs vaccine使用賽威FreeMix佐劑制備PCV3 VLPs疫苗，PCV3抗體在首次The PCV3 VLPs vaccine was prepared using FreeMix adjuvant,and the PCV3 antibody was免免疫疫后后3周周抗抗體體轉轉陽陽率率和二二免免后后抗抗體體水水平平上上均均優優于于商商品品水水佐佐劑劑。Three weeks after immunization,the antibody positive

51、rate and antibody level after second immunization were better than those of commercial water adjuvant.5.1 佐佐劑劑篩篩選選5.1 Adjuvant ScreeningP I%0 w3 wS e c-2 wS ec-2 w02 0206 0504 08 01 0 0賽威FreeMix佐劑組FreeMix adjuvant group某商品水佐劑A Commercial Water Adjuvant含含陰陰、陽陽兩兩種種離離子子基基團團的的聚聚丙丙烯烯酸酸-聚聚賴賴氨氨酸酸納納米米顆顆粒粒Po

52、lyacrylic acid-polylysine nanoparticles containing anionic and cationic groups-聚聚丙丙烯烯酸酸聚聚賴賴氨氨酸酸Polyacrylic acid polylysine形形成成聚聚電電解解質質復復合合物物Formation of olyelectrolyte complexes微微流流控控技技術術Microfluidic technology納納米米顆顆粒粒Nanopart i c le膜膜乳乳化化系系統統制制備備納納米米顆顆粒粒Preparation of nanoparticles by membrane emul

53、sification systemTEM觀觀察察納納米米顆顆粒粒TEM observation of nanoparticles新新型型水水性性佐佐劑劑Free Mix：可吸附攜不同電荷抗原、生物兼容性好；含增強天然免疫的非殺病毒性植物萃取物和雙基團納米顆粒，能實現聯合免疫。New aqueous adjuvant Free Mix：It can adsorb antigens with different charges and has good biocompatibility;it contains non-virucidal plant extracts and double-grou

54、p nanoparticles that enhance natural immunity and can achieve combined immunity.不不同同佐佐劑劑免免疫疫后后阻阻斷斷ELISA檢檢測測PCV3抗抗體體水水平平Detection of PCV3 antibody level by blocking ELISA after immunization with different adjuvants一帶一路國際生豬產業大會5、PCV3 VLPs疫疫苗苗的的創創制制5.Development of PCV3 VLPs vaccine疫苗免疫仔豬、成年豬精神飲食體溫均無異常

55、；免疫部位無局部不良反應，組織切片無異常病理變化。There was no abnormality in the spirit,diet and body temperature of vaccine-immunized piglets and adult pigs;there was no local adverse reaction at the immune site,and there was no abnormal pathological change in tissue sections.疫苗免疫懷孕母豬和公豬，精神飲食體溫無異常，繁殖性能無影響。In pregnant sows

56、 and boars immunized with the vaccine,there is no abnormality in mental diet and body temperature,and there is no effect on reproductive performance.5.2 疫疫苗苗安安全全性性5.2 Vaccine safetyT m0 d7 d1 4 d14d3 8.03 8.53 9.03 9.54 0.04 0.50 1010 20 303超超劑劑量量免免疫疫成成年年豬豬體體溫溫Body temperature of overdose immunized

57、adult pigs-TM-TM-0 d7 d1 4 d14d3 8.53 9.03 9.54 0.04 0.50 1010 20 303超超劑劑量量免免疫疫仔仔豬豬體體溫溫Body temperature of overdose immunized piglets超超劑劑量量免免疫疫仔仔豬豬注注射射部部位位Injection site of piglets with overdose immunization超超劑劑量量免免疫疫成成年年豬豬注注射射部部位位Injection site of adult pigs with overdose immunization一帶一路國際生豬產業大會5、

58、PCV3 VLPs疫疫苗苗的的創創制制5.Development of PCV3 VLPs vaccinePCV3 VLPs疫苗免免疫疫后后3周周抗抗體體開開始始轉轉陽陽；二二免免后后可產生高高水水平平的的PCV3抗抗體體。PCV3 VLPs vaccine Antibodies begin to turn positive 3 weeks after immunization;After the second immunization High levels of PCV3 antibodiescan be produced.5.3 疫疫苗苗效效力力5.3 Vaccine efficacy疫

59、苗免疫后抗體水平Antibody level after vaccination制備的3批疫苗免疫仔豬，二次免疫后抗抗體體可可持持續續至至少少4個個月月。The three batches of vaccines prepared were used to immunize piglets.After the second immunization,antibody lasts for at least 4 months.P I%0 w3 wS e c-2 wS ec-2 w02 0206 0504 08 01 0 0高劑量免疫組High dose immunization group低劑量免

60、疫組Low dose immunization groupP I%0 w3 wS e c-2 wS ec-2 wS e c-1 mS ec-1 mS e c-2 mS ec-2 mS e c-3 mS ec-3 mS e c-4 mS ec-4 m02 0206 0504 08 01 0 001批Batch 0102批Batch 0203批Batch 03疫苗免疫持續期Duration of vaccine immunity一帶一路國際生豬產業大會5、PCV3 VLPs疫疫苗苗的的創創制制5.Development of PCV3 VLPs vaccine5.3 疫疫苗苗效效力力5.3 Vac

61、cine efficacy攻攻毒毒后后平平均均日日增增重重Average daily gain after challengeA v e ra g ew e ig h t-g/dW e ig h t-g/d1 5 02 0 02 5 03 0 03 5 04 0 0高劑量免疫組低劑量免疫組空白對照組 攻毒對照組高劑量免疫組High dose immunization group低劑量免疫組Low dose immunization group 疫苗免疫后攻毒，平平均均日日增增重重與與正正常常對對照照豬豬一一致致，顯著高于攻毒對照；可阻阻斷斷PCV3產生病病毒毒血血癥癥和阻阻斷斷PCV3產生組組

62、織織帶帶毒毒。Challenge after vaccination,the average daily weight gain is consistent with normal control pigs,significantly higher than the challenge control;can block PCV3 generating Viremiaand toxic tissue.攻攻毒毒后后病病毒毒血血癥癥Viraemia post challengeC T V a lu e1 52 0202 53 03 54 0攻攻0d 14d21d 28d 攻攻0d 14d 21d

63、28d攻攻0d 14d 21d 28dChallenge 0d 14d 21d 28d Challenge 0d 14d 21d 28dChallenge 0d 14d 21d 28dC T V a lu e01 02 0203 04 0攻攻毒毒后后組組織織帶帶毒毒Virus-carrying tissues after challenge高劑量免疫組High dose immunization group低劑量免疫組Low dose immunization group攻毒對照組 Challenge control group肺臟Lungs肺門淋巴結Pulmonary hilar lymph

64、 node下頜淋巴結Mandibular lymph nodeHigh-dose immune Low-dose immune Blank control group Challenge control group一帶一路國際生豬產業大會PEDV亞亞單單位位疫疫苗苗的的創創制制Development of PEDV Subunit Vaccine二二一帶一路國際生豬產業大會主主要要內內容容Main contents2.S蛋蛋白白表表達達策策略略優優化化2.Optimization of S protein expression strategy3.S蛋蛋白白制制備備工工藝藝開開發發3.Prep

65、aration process development of S protein1.PEDV概概述述1.Overview of PEDVPEDV病病原原學學分分析析PEDV etiology analysisPEDV流流行行現現狀狀Epidemic status of PEDV抗抗原原分分析析Antigen analysis抗抗原原構構建建策策略略設設計計Antigen construction strategy design抗抗原原構構建建策策略略篩篩選選Screening of antigen construction strategy穩穩轉轉細細胞胞株株的的構構建建Constructio

66、n of stably transfected cell lines穩穩轉轉細細胞胞株株的的篩篩選選與與鑒鑒定定Screening and Identification of Stably Transformed Cell Lines表表達達工工藝藝開開發發Expression Process Development純純化化工工藝藝開開發發Purification process development4.PEDV亞亞單單位位疫疫苗苗創創制制4.PEDV subunit vaccine development疫疫苗苗安安全全性性Vaccine safety疫疫苗苗效效力力Vaccine eff

67、icacy一帶一路國際生豬產業大會1.PEDV概概述述1.Overview of PEDV病病原原：豬流行性腹瀉病毒（PEDV）屬于冠冠狀狀病病毒毒科、冠狀病毒屬，通過消消化化道道傳播（糞-口）。Pathogen:Porcine epidemic diarrhea virus(PEDV)belongs to Coronavirusfamily,genus Coronavirus,spread through digestive tract(fecal-oral).臨臨床床表表現現和和危危害害：主要表現為仔豬嘔嘔吐吐、嚴嚴重重腹腹瀉瀉和和脫脫水水；各年齡段的豬均可發病，但對哺哺乳乳仔仔豬豬的的危

68、危害害最最為為嚴嚴重重，20日齡以內仔豬的死亡率達95%以上。Clinical manifestations and hazards:mainly manifestations are vomiting,severe diarrhea,and dehydrationin piglets;Pigs of all ages can be affected,but Suckling piglets are most seriously affectedThe mortality rate of piglets within 20 days of age is over 95%.病病理理損損傷傷：主要

69、表現在豬的空腸、回腸部分的腸腸絨絨毛毛萎萎縮縮和和脫脫落落。Pathological damage:mainly manifested in atrophy and loss of intestinal villi in the jejunum and ileum of pigs.一帶一路國際生豬產業大會1.PEDV概概述述1.Overview of PEDV70.00%60.00%50.00%40.00%30.00%20.00%10.00%0.00%陽性率Positive rate2020年-2024年3月豬腹瀉病毒監測情況Porcine diarrhea virus surveillanc

70、e from 2020 to March 20242020年20202022年20222021年20212023年20232024年3月底End of March 20242020-2024.3月臨床腹瀉樣品來源Source of clinical diarrhea samples in March 2020-2024國家中心2020年-2024年3月豬病毒性腹瀉監測數據顯示：PEDV陽陽性性率率最最高高，其次是PRoV和PDCoV；PEDV和和PRoV混混合合感感染染率率高高。The National Centers swine viral diarrhea monitoring data

71、from 2020 to March 2024 showed:PEDV has the highest positive rate,followed by PRoV and PDCoV;The mixed infection rate of PEDV and PRoV was high.海海南南Hainan黑黑龍龍江江Heilongjiang吉吉林林Jilin遼遼寧寧Liaoning福福建建Fujian江江西西Jiangxi安安徽徽Anhui湖湖北北Hubei湖湖南南Hunan廣廣東東Guangdong廣廣西西Guangxi上上海海Shanghai河河南南Henan山山西西Shanxi內內蒙蒙

72、古古Inner Mongolia陜陜西西Shaanxi寧寧夏夏Ningxia甘甘肅肅Gansu四四川川Sichuan貴貴州州Guizhou云云南南Yunnan西西藏藏Tibet新新疆疆Xinjiang江江蘇蘇Jiangsu浙浙江江Zhejiang北北京京Beijing天天津津Tianjin河河北北Hebei山山東東Shandong青青海海Qinghai100 100-499500-900900送樣量Sample quantity delivered一帶一路國際生豬產業大會1.PEDV概概述述1.Overview of PEDV來源Source年份YearPEDV流行情況Prevalence o

73、f PEDV中科基因Zhongke Gene202235.37%科前Ke Qian2019-2022以PEDV為主Mainly PEDV齊魯Qilu202037.5%,GII型為主37.5%,mainly GII type天津瑞普Tianjin Ruipu2020-202350.70%，以GII為主50.70%,mainly GII溫氏Wens2021-202211.80%青島易邦Qingdao Yibang202310.80%，以GIIb為主10.80%,mainly GIIb山東綠都Shandong Lvdu2020-202240.20%河北銘諸生物Hebei Mingzhu Biolog

74、y202222.82%河南農大Henan Agricultural University2020-202135.2%，G2b為主35.2%,mainly G2b（數據來源：參考文獻或公眾號）(Data source:References or official accounts)S INDEL 7%0%2020-2024年年3月月PEDV不不同同基基因因型型分分布布Distribution of different genotypes of PEDV from 2020 to March 2024GIGIIS INDELGIIGIS INDEL GII GI國家中心2020年-2024年3月監

75、測數據，及其他9家單位監測數據顯示：GII型型為為主主要要流流行行的的PEDV毒毒株株。The monitoring data of the National Center from 2020 to March 2024 and the monitoring data of 9 other units show:GII type is the main prevalent PEDV strain.（數據來源：國家獸用藥品工程技術研究中心）(Data source:National Engineering and Technology Research Center for Veterinary

76、 Drugs)一帶一路國際生豬產業大會1.PEDV概概述述1.Overview of PEDV自自1978年年PED爆爆發發以以來來，毒毒株株在在不不斷斷的的變變異異Since the PED outbreak in 1978,the strain has been constantly mutatingPEDV S基因遺傳變異快，2010年PEDV的再次暴發，G2型毒力強，不斷給臨床帶來挑戰。The rapid genetic variation of PEDV S gene,the re-outbreak of PEDV in 2010,and the strong virulence o

77、f G2 continue to bring challenges to clinical practice.高高滴滴度度中中和和抗抗體體可有效控制PEDV在臨床上造成的危害。High titer neutralizing antibodies can effectively control the harm caused by PEDV in clinical practice.G1G1b1978 年年In 1978,CV 7 7 7比比利利時時BelgiumG1a1998 年年 SM 9 81998 SM 9 8韓韓國國Korea2003 年年 DR 1 32003 DR 1 3韓韓國國K

78、oreaG2G2b2006 年年 LZC2006 by LZC中中國國China由由S蛋蛋白白N端端差差異異決決定定區區分分Determined by the difference in the N-terminus of the S proteinG2a2010-2012 年年2010-20122010-2012 年年2010-2012AJ 11 0 2 LC 中中國國ChinaA H 2 0 1 2 GD-B中中國國ChinaS INDEL2013 年年 O H 5 8 12013 O H 5 8 1中中國國China重重組組Recombination2014 年年 CH-H NYF-14

79、 2014 CH-H NYF-14 中中國國China一帶一路國際生豬產業大會2、S蛋蛋白白表表達達策策略略優優化化2.Optimization of S protein expression strategy2.1 抗抗原原分分析析2.1 Antigen analysis通通過過對對PEDV S蛋蛋白白功功能能域域/中中和和表表位位的的分分析析，基基于于其其與與2019-nCoV S蛋蛋白白構構象象的的一一致致性性，確確定定優優勢勢抗抗原原區區域域。Through the analysis of PEDV S protein functional domain/neutralizing ep

80、itope and based on its similarity to 2019-nCoV S protein Conformational consistency,Predominant antigenic region is determined.Doi.org/10.1016/j.micpath.2020.104553PEDV與與新新冠冠S蛋蛋白白構構象象比比對對Conformation alignment of PEDV and 2019-nCoV S proteinPEDV S蛋蛋白白功功能能域域/中中和和表表位位分分析析Domain/neutralizing epitope an

81、alysis of PEDV S protein一帶一路國際生豬產業大會參參考考新新冠冠病病毒毒二二聚聚體體/三三聚聚體體/納納米米顆顆粒粒疫疫苗苗策策略略，形形成成PEDV S蛋蛋白白抗抗原原開開發發策策略略庫庫，獲獲得得不不同同聚聚集集形形式式的的S蛋蛋白白。Refer to the new coronavirus dimer/trimer/nanoparticle vaccine strategy to form PEDV S protein antigen development strategy library,obtaining S proteins in different ag

82、gregation forms.Zhang.Monoclonal Antibodies in Immunodiagnosis and Immunotherapy.February 2016,Vol.35,No.1:37-40.Xiao.Front Microbiol.2022 Oct 3;13:1018748.Huang.Animals(Basel).2022 Dec 1;12(23):3388.輔輔助助標標簽簽篩篩選選Secondary Label Filter不不同同聚聚集集體體形形式式的的蛋蛋白白Proteins in different aggregate formsS1S2S2S1S

83、1S1(主要抗原區)S1(major antigenic region)NTDCTDNTDCTDNTD(高重組區)NTD(region of high recombination)CTD(核心表位區)CTD(Core Epitope Region)NTD-CTD2.2抗抗原原構構建建策策略略設設計計2.2Antigen construction strategy design抗抗原原結結構構域域篩篩選選Antigen domain screening1138326S胞外域S extracellular domain1322267372624026506640240 5066402、S蛋蛋白白表

84、表達達策策略略優優化化2.Optimization of S protein expression strategy一帶一路國際生豬產業大會經經本本動動物物豬豬免免疫疫評評價價篩篩選選獲獲得得可可誘誘導導較較高高中中和和抗抗體體的的最最優優保保護護性性抗抗原原開開發發策策略略，二二免免后后中中和和抗抗體體效效價價顯顯著著提提升升：均均值值可可到到1:400。The optimal protective antigen development strategy for inducing higher neutralizing antibodies was obtained from the im

85、munization evaluation of this animal pig,and the neutralizing antibody potency was significantly increased after the second immunization:the mean value could reach 1:400.2.2 抗抗原原構構建建策策略略篩篩選選2.2 Screening of antigen construction strategy2、S蛋蛋白白表表達達策策略略優優化化2.Optimization of S protein expression strate

86、gyP E D V 不不 同同 策策 略略 中中 和和 抗抗 體體 比比 較較Comparison of neutralizing antibodies in different PEDV strategiesd p i中中 和和 效效 價價（1：n)Neutralizing potency(1:n)免免 3 WImmunization3 W二二 免免 1 WSecond Immunization 1 W1 6416 41 0 2 44 0 02 5 64 0 9 6策 略 1Strategy 1策 略 2Strategy 2策 略 3Strategy 3策 略 4Strategy 4策 略

87、5Strategy 5一帶一路國際生豬產業大會蛋白正確折疊&分泌水平提升Correct protein folding&increased secretion原始CHO細胞Primary CHO cellCas9技術構建過表達細胞系Construction of overexpression cell lines by Cas9 technologySNARE轉運蛋白&SNARE Transporters&分子伴侶Molecular chaperones篩篩選選基基因因Screening gene啟啟動動子子Promoter目目的的基基因因Target gene代謝類Metabolism抗生素

88、類Antibiotics病毒來源Source of Virus人延伸因子源Human elongation factor-derived雜合型啟動子 heterozygous promoterCHO宿宿主主細細胞胞的的基基因因工工程程改改造造Genetic Engineering of CHO Host Cells2.3 穩穩轉轉細細胞胞株株的的構構建建2.3 Construction of stably transfected cell lines表表達達載載體體核核心心元元件件的的優優化化Optimization of Core Elements of Expression Vector

89、優優選選與與宿宿主主細細胞胞匹匹配配的的篩篩選選基基因因、啟啟動動子子實實現現重重組組蛋蛋白白的的高高效效表表達達。Preferred screening genes and promoters matched with host cells to achieve efficient expression of recombinant proteins.CHO宿宿主主細細胞胞過過表表達達輔輔助助折折疊疊的的分分子子伴伴侶侶與與囊囊泡泡轉轉運運蛋蛋白白，抑抑制制未未折折 疊疊蛋蛋白白生生成成的的同同時時提提升升蛋蛋白白分分泌泌表表達達水水平平。CHO host cells overexpress

90、 molecular chaperones and vesicle transporters that assist folding,which inhibits the production of unfolded proteins and improves the secretion and expression level of proteins.2、S蛋蛋白白表表達達策策略略優優化化2.Optimization of S protein expression strategy一帶一路國際生豬產業大會123456789101112A B CD E F G H2.4 穩穩轉轉細細胞胞株株的

91、的篩篩選選與與鑒鑒定定2.4 Screening and Identification of Stably Transformed Cell Lines流流式式細細胞胞術術高高效效篩篩選選細細胞胞株株Efficient Screening of Cell Lines by Flow Cytometry細細胞胞株株的的穩穩定定性性鑒鑒定定Stability identification of cell lines親和捕獲法高效分選表達S蛋白的CHO細胞Efficient sorting of CHO cells expressing S protein by affinity capture (

92、1 0 5)%03691 21 51 82 12 42 73 03 33 63 94 24 54 85 15 45 76 06 36 66 97 27 57 88 18 42 0201 81 61 41 21 0864201 0 09 08 07 06 05 04 03 02 0201 00活 率Viability細 胞 數 目Number of cells細胞株倍增時間無明顯變化、克隆生長穩定No significant change in doubling time of cell lines,stable clonal growth通通過過親親和和捕捕獲獲法法高高效效篩篩選選表表達達PE

93、DV-S蛋蛋白白抗抗原原的的細細胞胞株株；細細胞胞株株在在30代代之之內內生生長長及及S基基因因遺遺傳傳穩穩定定。Use affinity capturefor efficient screening of cell lines expressing PEDV-S protein antigen;cell lines grow within 30 generations and the S gene is genetically stable.拷貝數變異20%Copy number variation 20%不不 同同 代代 次次 細細 胞胞 株株Strains of different ge

94、nerationsC T 值值C T valueP 5P 1 0P 2 0P 3 02 0202 12 22 32 42 52、S蛋蛋白白表表達達策策略略優優化化2.Optimization of S protein expression strategyP10P20P30P10 P20 P30P30代之內表達量均能超過1g/LThe expression amount can exceed 1 g/L within P30 generation流式細胞儀分選Flow cytometry sorting一帶一路國際生豬產業大會從從細細胞胞株株特特性性、培培養養過過程程調調控控及及產產物物構構象

95、象反反饋饋，建建立立完完善善的的PEDV-S蛋蛋白白抗抗原原高高效效表表達達工工藝藝。Establish a perfect process for efficient expression of PEDV-S protein antigen from cell strain characterization,culture process regulation and product conformation feedback.產產物物構構象象反反饋饋調調節節梯梯度度溫溫度度培培養養工工藝藝Gradient temperature culture process with product c

96、onformation feedback regulation3、S蛋蛋白白制制備備工工藝藝開開發發3.S protein preparation process development3.1 S蛋蛋白白表表達達工工藝藝開開發發3.1 S protein expression process development工工藝藝優優化化Process optimization結合細胞生理狀態及能量代謝流調控，建立以滲透壓為標準的補料工藝；Establish a feeding process with osmotic pressure as the standard in combination w

97、ith the physiological state of cells and the regulation of energy metabolic flow;挖掘細胞株微量元素需求特異性，針對性添加“亞鐵離子”開關調控；Explore the specificity of trace element requirements of cell lines,and add ferrous ion switch regulation in a targeted manner;依據細胞生長動力學及產物構象反饋，選擇梯度溫度培養策略，同時滿足目標蛋白的“量”和“質”。According to the

98、 cell growth kinetics and product conformation feedback,the gradient temperature culture strategy was selected to meet the quantity and quality of the target protein.一帶一路國際生豬產業大會3、S蛋蛋白白制制備備工工藝藝開開發發3.S protein preparation process development3.1 S蛋蛋白白表表達達工工藝藝開開發發3.1 S protein expression process develo

99、pment工工藝藝放放大大Process scale-up通過糖酵解通量調節及代謝過程關鍵酶活控制，維持細胞內氧化還原平衡穩態，有效提高蛋白表達效率。By regulating glycolytic flux and controlling key enzyme activities in the metabolic process,it maintains the homeostasis of intracellular redox balance and effectively improves the efficiency of protein expression.計算反應器上細胞培養

100、過程的計量學和動力學，通過縮小模型驗證，實現150L反應器的有效放大。Calculate the metrology and kinetics of the cell culture process on the reactor,and achieve the effective scale-up of the 150L reactor through the verification of the scaled-down model.采采用用優優化化的的工工藝藝參參數數，進進一一步步調調節節反反應應器器上上糖糖酵酵解解通通量量及及代代謝謝過過程程關關鍵鍵酶酶活活，實實現現150L反反應應器器

101、的的有有效效放放大大。Use optimized process parameters to further adjust the reactor glycolytic fluxand key enzyme activity in metabolic processes,achieving efficient Scale-up of 150L Reactor.一帶一路國際生豬產業大會結結合合超超濾濾與與層層析析純純化化技技術術，建建立立有有效效抗抗原原純純化化工工藝藝：高高效效分分子子排排阻阻色色譜譜(HPSEC)檢檢測測S蛋蛋白白為為構構象象穩穩定定、單單一一的的寡寡聚聚物物。Combine

102、 ultrafiltration and chromatography purification technology to establish an effective antigen purification process:High performance size exclusion chromatography(HPSEC)detection of S protein as conformation stable,single oligomers.3、S蛋蛋白白制制備備工工藝藝開開發發3.S protein preparation process development3.2 S蛋蛋

103、白白純純化化工工藝藝開開發發3.2 S protein purification process development收收獲獲澄澄清清Harvest Clarification切切向向流流超超濾濾Tangential flow ultrafiltration層層析析純純化化Chromatographic purification批次1 批次2 批次3Batch 1 Batch 2 Batch 3穩穩定定性性監監測測Stability monitoring一帶一路國際生豬產業大會4、PEDV 亞亞單單位位疫疫苗苗的的制制備備4.Preparation of PEDV subunit vacci

104、neT m0 d1 d2 d3 d4 d5 d6 d7 d8 d9 d1 0 d1 1 d1 2 d1 3 d1 4 d14d3 8.53 9.03 9.54 0.04 0.5C o n 0 30 20 101-C疫苗免疫仔豬、成年豬精神飲食體溫均無異常，免疫部位無異常。The spirit,diet and body temperature of vaccine-immunized piglets and adult pigs were normal,and there was no abnormality in the immune site.疫苗免疫懷孕母豬，精神飲食體溫無異常，生產成績

105、不受影響。The pregnant sows immunized with the vaccine have no abnormalities in mental,diet and body temperature,and the production performance is not affected.4.1 疫疫苗苗安安全全性性4.1 Vaccine safety一帶一路國際生豬產業大會4、PEDV 亞亞單單位位疫疫苗苗的的制制備備4.Preparation of PEDV subunit vaccine4.2 疫疫苗苗效效力力仔仔豬豬4.2 Vaccine efficacy-pigl

106、ets初生仔豬免疫一次，斷奶后攻毒評價：免疫仔豬攻毒后的日日平平均均采采食食量量和增增重重情情況況顯顯著著優優于于攻毒對照組；Newborn piglets were immunized once and challenged with virus after weaning:Average daily feed intakeand the weight gain was significantly better thanthe challenge control group;斷奶仔豬攻毒保護情況：攻攻毒毒對對照照組組5/5腹腹瀉瀉。免免疫疫組組5/5保保護護。Protection of we

107、aned piglets from virus attack:5/5 of the challenge control group had diarrhea.5/5 of the immunized group were protected.fo o dFo o dc o n s u m p tio n-gC o n s u m p tio n-g1 d2 d3 d4 d5 d6 d7 d02 0 04 0 06 0 08 0 01 0 0 0攻毒對照組Challenge control group免疫組Immunization group攻毒后仔豬采食量Feed intake of pigl

108、ets after virus challengeA v e ra g e w e ig h t-g/d-0.1 5-0.1 0-0.0 50.0 00.0 50.1 0免疫組Immunization group攻毒對照組Challenge control group攻毒后仔豬日增重Daily gain of piglets after virus challenge一帶一路國際生豬產業大會4、PEDV 亞亞單單位位疫疫苗苗的的制制備備4.Preparation of PEDV subunit vaccine4.3 疫疫苗苗效效力力母母豬豬免免疫疫后后抗抗體體和和被被動動免免疫疫保保護護4.3

109、 Vaccine efficacy-antibodies and passive immune protection after sow immunization母豬分娩前免疫兩次，分娩仔豬，可從從初初乳乳中中可可獲獲得得高高水水平平的的中中和和抗抗體體，仔仔豬豬獲獲得得高高水水平平中中和和抗抗體體至至出出生生后后28日日。Sows are vaccinated twice before farrowing and piglets are born.High levels of neutralizing antibodies can be obtained from colostrum.Pig

110、lets acquire high levels of neutralizing antibodies until 28 days after birth.A 1 5 3A 15 3A 2 2 1 6 7A 22 1 6 7Y 0 0 0 1Y 00 0 1C o nCon0123456保護數Number of protection（頭Head）分娩仔豬在24-26日齡攻毒，對對照照組組豬豬5/5腹腹瀉瀉，Piglets are challenged with the virus at 24-26 days of age.5/5 pigs in the control group had di

111、arrhea.試試驗驗仔仔豬豬（斷斷奶奶）5/5保保護護。Test piglets(weaned)5/5 protection.一帶一路國際生豬產業大會4、PEDV 亞亞單單位位疫疫苗苗的的制制備備4.Preparation of PEDV subunit vaccine4.3 疫疫苗苗效效力力免免疫疫持持續續期期4.3 Vaccine Efficacy-Duration of Immunity一次免疫后3周，PEDV亞單位疫苗中和抗體4/51：16；At 3 weeks after the first immunization,the neutralizing antibody of PED

112、V subunit vaccine is 4/5 1:16;二次免疫后中和抗體效價顯著提升，二二免免后后1-5周周中中和和抗抗體體效效價價均均值值可可到到1:400以以上上；After the second immunization,the neutralizing antibody titer increased significantly.The average neutralizing antibody titer can reach above 1:400 1-5 weeks after the second vaccination;PEDV亞單位疫苗二免后，高水平中中和和抗抗體體可可

113、持持續續至至二二免免后后4個個月月。After the second immunization with PEDV subunit vaccine,high levels Neutralizing antibodies can last up to 4 months after the second vaccination.V N(1/n)1 w2 w3 w2-1 w2-1 w2-2 w2-2 w2-3 w2-3 w2-4 w2-4 w2-5 w2-5 w2-6 w2-6 w2-8 w2-8 w2-1 2 w2-1 2 w2-1 6 w2-1 6 w146 41 0 2 44 0 02 5 64 0 9 616疫苗免疫持續期中和抗體水平檢測Detection of neutralizing antibody level during the duration of vaccine immunity一帶一路國際生豬產業大會感感謝謝蒞蒞臨臨國國家家獸獸用用藥藥品品工工程程技技術術研研究究中中心心指指導導！Thank you for coming to the National Engineering Research Center for Veterinary Drugs for guidance!一帶一路國際生豬產業大會