Preparation,Identification And Biological Function Of MAbs Against Glaesserella Parasuis

Glaesserella parasuis（G.parasuis）,once called Haemophilus parasuis,is the causative agent of Gl?sser’s disease of swine.The disease seriously hampers the health of postweaning pigs.The current prevention and treatment measures for Gl?sser’s disease mainly include antibiotic treatment and vaccination.However,the abuse of antibiotics not only leads to a significant increase in the resistance of bacteria,but also leads to drug residues,which ultimately endanger human health.In the context of restricting the use of antibiotics in China,vaccination has become the best measures to prevent and treat Gl?sser’s disease.But there are many serovars of G.parasuis,and a large proportion of them cannot be typed.This leads to the weak cross-protection of existing whole-bacteria vaccines between strains of different serovars.It is particularly important to expand new vaccine research strategies.Monoclonal antibody（MAb）is an indispensable tool in basic research,disease prevention and treatment,and has a high degree of sensitivity and specificity.The acquisition of functional MAb can not only lay a theoretical foundation for screening the dominant epitopes of G.parasuis,but also provide a strong material guarantee for the prevention and treatment of Gl?sser’s disease.Antigen-antibody complex vaccine is an emerging vaccine,which has been successfully applied to the immunization of infectious bursal disease and Newcastle disease in chickens and achieved good results.There has been no report on the application of antigen-antibody complex vaccines in the prevention of Gl?sser’s disease.Based on the above research background,this article has conducted the following researches.1.Preparation of MAbs against G.parasuis serovar 5 strain and identification of its target proteinIn this study,G.parasuis standard serovars 5 strain kept in our laboratory were inactivated by formaldehyde to prepare a whole bacterial suspension.After emulsified with freund’s adjuvant,seven BALB/c mice were immunized by multiple subcutaneous injections on the back.Six positive hybridoma cell lines that can stably secrete antibodies were screened through traditional monoclonal antibody preparation technology,and they were named 3E3,4B2,1A12,2D1,3E6,and 4C6.The MAb 3E3,4B2,1A12,2D1,3E6,and 4C6 were injected intraperitoneally into mice to prepare ascites.The subtype identification results showed that MAb 3E3,4B2,1A12,2D1,3E6,4C6 are all Ig M subtypes,κchain.The results of the indirect ELISA test showed that the ascites titer of the3 monoclonal antibodies 4B2,1A12,and 2D1 reached 1:51200.The results of laser confocal test and flow cytometry analysis showed that these six MAbs could all react with the G.parasuis serovar 5 standard strain.The indirect ELISA test showed that MAb 2D1can react with the 1～15 serovars of standard strains of G.parasuis,so MAb 2D1 was selected for subsequent tests.The size of the target protein of MAb 2D1 was determined to be between 35～40 k Da by western blot.The target location on the SDS-PAGE was cut and analyzed.Three possible target proteins were selected by mass spectrometry analysis:Fe³⁺ABC transporter substrate binding protein,porin and GAPDH.The three proteins were successfully expressed in prokaryotic cells.Finally,it was determined by western blot that the target protein of MAb 2D1 was Fe³⁺ABC transporter substrate binding protein.After consulting the relevant literature,it is found that Fe³⁺ABC transporter substrate binding protein is related to the mechanism of bacteria uptake of iron.The cross-reactivity between MAb 2D1 and 1～15 serovars standard strains of G.parasuis indicated that Fe³⁺ABC transporter substrate binding protein exists in 15 serovars strains of G.parasuis.This suggests that the protein is likely to be a potential immunodominant antigen of G.parasuis,which lay a theoretical foundation for the study of subunit vaccines with good cross-protection.2.Functional research of MAb 2D1The functionalities of MAb 2D1 were investigated from the aspect of in vitro and in vivo.In all tests,rabbits polyclonal antibody against G.parasuis serovar 5 standard strain（positive serum prepared by immunizing rabbits preserved in the laboratory）was set as a positive control,PBS and the laboratory’s existing MAb against Apx IV of Actinobacillus pleuropneumoniae（APP）were used as the negative control,at the same time,a blank control was used.In vitro tests include complement killing experiment and opsonized phagocytosis tests.The results of the complement killing experiment showed that the number of colonies of the G.parasuis serovar 5 standard strain treated with MAb 2D1 was significantly less than that of the negative control group（P<0.01）,indicating that MAb 2D1can activate the complement killing to inhibit the growth of G.parasuis.In the opsonization assay,3D4/21 cells can significantly improve the phagocytic ability to the standard strain of G.parasuis serovar 5 pretreated with MAb 2D1（P<0.05）.When RAW264.7 cells with stronger phagocytic ability were used for the same opsonization phagocytosis test,the difference became more obvious（P<0.01）.This indicates that MAb 2D1 has oposonization effect and can enhance the immune clearance of phagocytes against the standard strain of G.parasuis serovar 5.In the passive immune protection test in mice,the survival rate of passively immunized MAb 2D1 and positive serum mice was 100%.At 48 h after challenge,the bacteria in the blood of these two groups of mice could be completely eliminated.The survival rate of mice passively immunized with MAb Apx IV was 40%.At 12,24,48,and72 h after challenge,the mice in this group all showed strong levels of bacteremia.The above results show that MAb 2D1 has complement killing effect and oposonization effect.In the mouse model,MAb 2D1 can resist the attack of the standard strain of G.parasuis serovar 5 to a certain extent and has the ability to eliminate bacteria.This indicates that MAb 2D1 is a high-potency antibacterial antibody,which has the potential to be developed as a biological preparation for the prevention and treatment of G.parasuis.It also further suggests that the protein targeted by MAb 2D1 has good immunogenicity.3.Evaluation of the protection of antigen-antibody complex vaccinesIn this study,40 ICR mice were selected and randomly divided into 5 groups,each with 8 mice,which were set as the inactivated vaccine group,the antigen-antibody complex vaccine group,the negative control group,the challenge control group,and the blank group.The prepared vaccine was inoculated into mice,and sera of mice were collected before the first immunization,two weeks after the first immunization,and two weeks after the secondary immunization to detect the antibody level by indirect ELISA method.Two weeks after the secondary immunization,three mice in each group were euthanized to isolate peripheral blood lymphocytes,and the total RNA was extracted for reverse transcription.The cytokine levels of mice were detected by SYBR Green real-time fluorescence quantitative PCR.Then,the mice were challenged and observed for 7 days.Micropathological changes of lungs and spleens of mice in each group were observed.The results showed that TNF-α,IL-6,IFN-γcytokine levels in the antigen-antibody complex vaccine group were not significantly different than those in the inactivated vaccine group and the negative control group（P>0.05）.However,IL-10 cytokine levels were significantly lower than those in the two groups（P<0.05）,and Ig G2a/Ig G1 value（0.7）of the antigen-antibody complex vaccine group was significantly higher than that of the inactivated vaccine group（0.54）and the negative control group（0.55）（P<0.05）,which suggest that antigen-antibody complex vaccine may maintain the Th1/Th2 balance immune response by down-regulating Th2 type response,and the vaccine can also reduce the inflammation of mice.The survival rates of both the inactivated vaccine group and the immune complex vaccine group were both 60%,and the pathological observation results showed that the pathological changes of the immune complex vaccine group on lungs were not significantly different from that of the inactivated vaccine group.The above results suggest that the antigen-antibody complex vaccine of G.parasuis standard serovar 5 strains has a good immune protection effect,which provides a new idea for the research and development of a new vaccine against G.parasuis.