| P.multocida(Pm)is A Gram-negative short bacillus with no flagellate,no spore formation and strong bipolar infection.It is one of the very important pathogens causing respiratory diseases and syndromes in cattle.It can be spanided into five kind of serotypes(A,B,D,E and F)in line with the sort of capsular antigens.Popular bovine sources Pm A:1,A:3,B:2,B:5 and E:2 are the main serotypes in foreign countries.In our country,before 2006 was mainly popular B:2,B:5 serum type bovine source Pm,since 2008,has successively separated from many regions across the country to domestic newly emerged bovine source type A Pm,and was in a popular trend,causing livestock husbandry industry to suffer significant economic losses.Currently,most of the vaccines against bovine pasteulosis in our country are inactivated vaccines against type B Pm.However,inactivated vaccines can not maintain immunity and between different Pm cross protection is poor,so it is vital to develop vaccines which have good immune protection against type A and type B Pm.In the previous laboratory study,two gene deletion strains were obtained,and the inactivated vaccines of both strains could achieve 100%protection against type A Pm and 87.5%-100%protection against type B Pm.High throughput transcriptomic sequencing was performed on the two gene deletion strains and their wild strains.Combined with transcriptome data analysis of the two gene deletion strains,supposed that the gene deletion influenced the expression of immune protective epitopes,so that the inactivated vaccine of the missing strain had better cross immune protection.Therefore,in this study,the upregulated differential genes shared by the two deletion strains were compared,and the secreted proteins,outer membrane proteins,virulence factors and unknown proteins with abundant antigen epitopes were focused on,combined with reverse vaccinology strategy and bioinformatics analysis,in order to screen out vaccine antigen targets with good cross-immune protection against both type A and type B Pm.The results were verified by the immunological protection tests of single-component antigen,two-component antigen and fusion expressed antigen,which offered foundation of novel subunit vaccines with higher safety and better immunogenicity.The prime research means and consequences are as follows:1.Selecting,cloning and expression of protective genes of Pasteurella multocida type A and purification of expression productsIn this study,altogether of 40 genes were selected based on the transcriptomic sequencing results of the two gene deletion strains in the previous laboratory study and combined with bioinformatics analysis.Specific primers were designed and amplified,construct and induced recombinant expression plasmid,then expressed products were purified.The consequences manifested that all 40 genes could be amplified correctly.All recombinant expression plasmids were constructed successfully.29 genes could be correctly expressed of all genes,1 gene was expressed but the size was not as expected(could not be correctly expressed after vector replacement),and 10 were not expressed.Among the 29 correctly expressed genes,10 had low expression,and the expression product of 1 gene was not bound to Ni column when purified.In this paper,18 antigen proteins were successfully purified in large quantities,and the purification effect was good,which could be used for immune protection test.2.Immunoprotective test of single-component subunitIn this study,18 antigenic proteins were successfully purified to prepare single-component subunit vaccine.Mice were immunized twice.Collected serum of mice after immuniting second,used ELISA test antibody titer assays.14 days after inoculation,mice were infected with Pm CQ2(type A)of 3.37×10~7 CFU to count the protection rate.Choosed the antigen with good protection to immunite mice again and infected with Pm B(type B)of 1×10~7 CFU to select the cross-protective proteins.The consequences showed that the lowest antibody titer of each protein was more than1:12800,indicating that the immunization effect of the vaccine was good.18 kinds of proteins played different immune protective effects on type A Pm,among which the titer assay of PM1009,PM1621,PM2065,PM1618,PM0811 was more than 1:102400at the lowest,the immune protective effect is good.The protection rate was 62.5%,50%,40%,30%and 30%in order.The five proteins also had a certain protective effect against type B Pm infection,with the lowest antibody titer above 1:102400,the protection rate was 75%,62.5%,42.9%,28.6%and 37.5%.Among which PM1618 had weak protective effect against type B Pm.In conclusion,PM1009,PM1621,PM2065and PM0811 all have good protective effects against type A and type B Pm,which might be potential cross-protective antigens.3.Immune protection test of two-component subunitIn order to explore whether the combined use of two antigens has additive synergistic effect,according to the results of the cross-protection test of single-component antigen proteins on Pm,we selected four proteins with the best cross-protection into four groups:PM1009+PM1621,PM1009+PM2065,PM1009+PM0811,PM1621+PM0811,and equal proportion of the mixture to immunize mice with two-component subunit vaccine.Collected serum of mice after immuniting second,used ELISA test antibody titer assays.14 days after inoculation,mice were infected with Pm CQ2(type A)of 3.37×10~7 CFU to count the protection rate.Choosed the antigen with good protection to immunite mice again and infected with Pm B(type B)of 1×10~7 CFU.The consequences indicated that the antibody levels of four combinations were all over 1:14800,indicating that the two-component vaccine could promote the production of higher levels of specific antibodies.The protection rates against type A Pm were 50%,50%,25%and 33.5%,respectively.Groups of PM1009+PM1621 and PM1009+PM2065 also had certain protective effects on type B Pm,with the protection rates of 50%and 37.5%,respectively.The two-component subunit vaccine provided only partial protection against Pm infection,presumably due to the halved amount of each protein when the vaccine was mixed.So we increased the immune dose of PM1009+PM1621 group with optimal cross protection(double the amount of both proteins,each 100μL vaccine contained each protein 100μg),collected serum of mice to determine antibody titer,and counted the protection rate by Pm CQ2 infection of 3.37×10~7 CFU on the 14th day after immunization.The consequences indicated that the level of antibody production was significantly different from lower dose group(**p<0.01),and the protection rate was62.5%,which was increased by 12.5%.These results indicated that within a certain immune dose,increasing the dose could promotr producing a higher level of antibody and improving the protection rate of mice,but it was still not enough to achieve 100%protection.4.Immunoprotective test of fusion proteinIt has been reported that fusion expression can synthesize the functions of multiple genes,improve the protection rate,and facilitate detection,separation and purification,which is an optimal expression strategy.Based on the results of single-component and two-component antigens,we found two proteins with the best cross-protection:PM009 and PM1621.Since the two proteins in this study were fully expressed and contained all epitopes,it is highly unlikely that they would have a better protective effect even if expressed by fusion.Therefore,in order to explore fusion antigens with better cross-protection,plp E gene and Pm1009 gene,which have been studied on immune protection at home and abroad,were selected for fusion expression,so as to provide data support for clinical research and development of fusion subunit vaccine.In this study,plp E and PM1009-plp E fusion proteins were expressed and prepared into subunit vaccines,and immunological protection tests were conducted against type A and type B Pm,respectively.The results showed that the protection rates of plp E protein against Pm CQ2 and Pm B were 50%and 25%,and the protection rates of PM1009-plp E against Pm CQ2 and Pm B were 75%and 50%,respectively.The protection rate of PM1009-plp E fusion protein against both type A and type B Pm infection was improved,suggesting that PM1009 protein may be a potential cross-protective antigen,and fusion expression is a feasible idea for future subunit vaccine development. |
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