Praperation Of Monoclonal Antibodies Against Pseudorabies Virus And Development Of Blocking ELISA For Detection Of GE Antibody

Pseudorabies(PR),also known as Aujeszky's Disease(AD),is a variety of animal comorbid infectious diseases caused by Pseudorabies virus(PRV).China is one of the high-incidence areas of PR.Since the introduction of the Bartha-K61 vaccine with gE gene deletion from Hungary in the 1970s,PR has been well controlled.However,due to the mutation of the virus,at the end of 2011,PR has broken in many pig farms with vaccination coverage,causing great economic loss to the swine industry of China.In this study,we first investigated the immune and infection status of PRV in East China.It was found that the immune status of pigs in East China was better,but the PRV wild-type infection was more serious,especially the sow herd.Secondly,15 hybridoma cell lines that could stably secrete the monoclonal antibody(McAb)against PRV were prepared using purified PRV ZJ01 virions and its gB,gC and gE proteins as immunogens.Then,the epitopes recognized by the McAbs were identificated by Western blot,and the conservation of epitopes and the characteristics and location of epitopes in the 3D structural model were analyzed.Finally,using one of the McAbs 3E1,a blocking ELISA method for detecting PRV gE antibodies was established.This study laid an important foundation for the prevention and control of the disease.The main contents of the research were as following:1.Epidemiological survey of porcine pseudorabies in East China in 2017In 2017,10179 serum samples were collected from 310 farms and tested by PRV gE-ELISA and gB-ELISA kits.The results showed that,the positive rate of gE and gB antibody at the farm level was 80.00%(248/310)and 100.00%(245/245)respectively,while at individual level it was 44.66%(4546/10179)and 95.90%(7600/7925).According to statistical analysis of infection rates in different province,it was found that the positive rate of serum samples in Shandong Province was highest(62.16%),and it was lower in Jiangsu(28.21%)and Anhui provinces(32.83%).According to statistical analysis of infection rates in different seasons,it was found that the positive rate was highest in spring(55.21%)and lowest in winter(29.38%)and summer(33.09%).Besides,the positive rate of PRV gE antibody in serum samples from different feeding stages was statistically found that the breeding sows had the highest positive rate,accounting for 60.59%,followed by multiparous sows(54.69%),and the breeding boars had the lowest rate(27.11%).As a result,the immune status of pigs in East China is better,and there is little difference between the provinces.But the infection status of PRV is serious,especially in Shandong Province.2.Preparation and identification of the monoclonal antibodies against pseudorabies VirusThe purified virions and proteins were used as immunogens,and 15 McAbs were obtained.Among them,2 hybridoma cell strains secret antibodies to gB,which were named 5A2 and 6G5 respectively.2 hybridoma cell strains secret antibodies to gC,which were named 5D10 and 7C5 respectively.6 hybridoma cell strains secrets antibody to gE,which were named 3E1,3H8,4D2,5E12,6H12 and 6E9 respectively.The other 5 McAbs were named 4B9,4F7,4C3,5E5 and 5E7.The cell culture supernatant ELISA antibody titer was 1:32?6400 and the ascites antibody titer was 1:6400?1024000.The results of antibody subtype identification showed that 5A2,6G5 and 5D10 belong to IgA;7C5 belong to IgG2b;3E1,3H8,4D2,5E12,6412,6E95 4C3 and 4F7 belong to IgG1;4B9,5E5 and 5E7 belong to IgG2a;and all the light chains are k type.Western blot and IFA results showed that 15 monoclonal antibodies could specifically react with PRV ZJ01.The development of these McAbs lays the foundation for the research of PRV functional proteins and the development of antibody diagnostic kits.3.The identification of B-cell epitopes of gB,gC and gE proteins of PRVA series of truncated recombinant gB,gC,and gE proteins were expressed in E.coli BL21.The epitopes recognized by the 10 McAbs were identified by Western blot.The resulted showed that the epitope recognized by the two McAbs against gB is located in 576aa?582aa of the gB.The epitope recognized by 5D10 McAb is located in 134aa?138aa of the gC,and the epitope recognized by 7C5 McAb is located in143aa?153aa of the gC.Six McAbs against the gE protein recognize the same epitope,located in 151aa?155aa of gE.In addition these linear B-cell epitopes were highly conserved among the reference strains.Analysis of the characteristics and localization of the above epitopes in the 3D structural model revealed that the epitopes 576SAVATAA582 and 151IGDYL155 form a part of a-helix respectively,and the epitopes 134GETFE138 and 143RRGRFRSPDAD153 exhibit a random coil.Only the epitopes 143RRGRFRSPDAD153 and 151IGDYL155 are completely exposed to the gC and gE protein surfaces,respectively,which may be the dominant antigenic epitopes of the proteins.4.Development of a blocking ELISA for detection of gE antibody to PRVA blocking ELISA method for detecting PRV gE antibody was established by using purified recombinant gE protein as a coating antigen and HRP-labeled 3E1 against gE.The optimized reaction conditions for each step were as following:Antigen coating concentration was 0.75?g/mL and coating at 4? for 12h;phosphate buffer containing 2%gelatin was used as a blocking agent,37? for 2 hours;the serum samples to be tested was not diluted and the incubation time was at 37? for 2h;the dilution of the enzyme standard monoclonal antibody was at 1:30000,and the incubation time was at 37? for 0.5h.This method was not cross-reactive with common swine disease antibody positive serum.The inter-batch and intra-assay coefficients of variation were all less than 12%.The sensitivity and specificity were 85.14%and 95.71%,respectively.The compliance rate with the IDEXX PRV gE antibody detection kit was 92.40%.Using this method,523 clinical serum samples were tested,and the positive rate of PRV gE antibody was 28.7%.Therefore,the blocking ELISA method can be used to identify PRV wild-type infection and vaccine immunity.In summary,we investigated the immune and infection status of PRV in East China,15 monoclonal antibodies were successfully prepared,the epitopes recognized by some monoclonal antibodies were identified,a blocking ELISA method for detecting PRV gE antibody was established using one of the McAbs.In conclusion,this study lays the foundation for the study of PRV protein structure and function,diagnosis of RR,epidemiological investigation and prevention and control of RR.