| Mucosal immunity plays a key role in maintaining local immune homeostasis of the body,and immunoglobulin A is the main effector molecule of mucosal immune response.In this study,the anti-Bactrian camel IgA antibody with high-efficiency and specific was prepared through molecular biology,immunohistochemistry and immunology.This will provide a basis for further investigation of the related immunological functions of Bactrian camel IgA,and lay a foundation for revealing the mucosa immune mechanism of Bactrian camel.The biological information of Bactrian camel IgA was analyzed firstly.The primary structure specificity of Bactrian camel IgA heavy chain constant region(IgA-H)was analyzed by software DNAMAN,and B cell epitopes of IgA-H was predicted by BepiPred 2.0.The best specific gene sequence(F-IgA-H)was screened by the results of the analysis.The physicochemical properties of IgA-H and F-IgA-H were analyzed by ProtParam.Secondly,prokaryotic expression plasmid pET-28a-IgA-H of IgA-H and prokaryotic expression plasmid pET-28a-F-IgA-H of F-IgA-H.These two plasmids were induced to express in BL21,meanwhile,the culture conditions were optimized,finally,the expression results were verified by SDS-PAGE and Western Blot.In addition,to verify the results of bioinformatics screening.The rabbit anti-Bactrian camel IgA-H polyclonal antibody(PAb-H)and rabbit anti-Bactrian camel F-IgA-H polyclonal antibodies(PAb-F)were prepared by purified recombinant protein respectively.Antibody titers and specificity were determined by ELISA and Western Blot.And then the IgA~+cell distribution characteristics in palatine tonsil was observed by SABC-immunohistochemistry staining(Primary antibodies were PAb-H,PAb-F and the polyclonal antibody of rabbit anti-Bactrian camel IgA(PAb-N)which was stored in the laboratory),then,the IgA~+cell density in different age groups were calculated by image-Pro Plus,and their differences were analyzed by SPSS 21.0 with one-way ANOVA.The results showed that:(1)Bioinformatics of Bactrian camel IgA was analyzed and a representative fragment F-IgA-H was selected from IgA-H.(2)Prokaryotic expression plasmid pET-28a-IgA-H of IgA-H and pET-28a-F-IgA-H of F-IgA-H were constructed,and the recombinant proteins were obtained from these plasmids.The results showed that recombinant protein IgA-H was mainly expressed in the form of inclusion bodies,the molecular weight was about 39 KD,and the optimal induction condition was IPTG0.1 mmol/L and the induction time was 6 h.The other recombinant protein F-IgA-H was expressed in the supernatant,the molecular weight was about 20 KD,the optimal induction condition was IPTG 0.15 mmol/L,and induction time 4 h.(3)The ELISA results showed that the titer of PAb-F(1:64000)was higher than PAb-H(1:32000),and the specific of two polyclonal antibodies was confirmed by Western Blot.(4)The results of immunohistochemistry showed that the working concentration of PAb-F(1:900)was higher than PAb-H(1:400).The difference analysis showed that there was no significant difference(P>0.05)in IgA~+cell density between left and right palatine tonsils in all age groups.The distribution density of IgA~+cells of the tonsils,in young group,there were significantly differences(P<0.05)between PAb-F and PAb-H and there was no significant difference(P>0.05)with PAb-F and PAb-N;in the pubertal group,there was significant difference between PAb-F,PAb-H and PAb-N(P<0.05);in the middle-aged group,the difference was significant(P<0.05)in PAb-F,PAb-H,and PAb-N.PAb-F prepared through the fragment F-IgA-H of IgA-specific epitope in Bactrian camel analyzed by bioinformatics,the titer and immunohistochemistry working concentration of PAb-F were higher than PAb-H and PAb-N,and the difference analysis of immunohistochemistry verification also showed that PAb-F was more efficient and specific than PAb-H and PAb-N.These results was consistent with bioinformatics analysis,and it would lay a foundation for revealing the mucosal immune mechanism of Bactrian camel. |
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