Preparation Of Different Immune Preparations Of Sheep Inhibin Alpha And Evaluation Of Its Immune Effect

Object: Xinjiang is one of China’s five pastoral areas,animal husbandry has a greater space for development.Kazak sheep is a local coarse wool sheep breed in Xinjiang,which is favored by local people for its excellent production performance and delicate meat quality.However,kazakh sheep’s low fertility and long breeding cycle make the supply of mutton far from meeting the market demand.Therefore,to explore new methods and techniques to improve the breeding efficiency of Kazakh sheep has become one of the main problems in xinjiang sheep breeding industry.Inhibin alpha can selectively inhibit the synthesis and secretion of follicle-stimulating hormone(FSH)through the endocrine negative feedback regulation pathway,thus affecting the feculence of animals.This study based on the characteristics of inhibin alpha(INH alpha)function,the use of bioinformatics,protein purification technology,antibody purification technology,the preparation of low cost,high immune titer of INH alpha antigen,using genetic immune method will INH alpha active and passive immunization Kazakh sheep,and study the effects of INH alpha on Kazakh sheep reproductive hormones and safety testing of the body,for INH alpha role in improving the efficiency of Xinjiang sheep breeding research mechanism to provide the reference data,but also provide new way for the preparation of camel INH alpha antibodies.Methods:(1)Optimize and identify the induced expression conditions of INH alpha recombinant plasmid prokaryotic expression strains,and collect the expressed proteins by nickel column affinity chromatography and urea dialysis renaturation,the protein was digested by enterokinase and purified by secondary purification,immunogenicity of the protein was identified by Western blotting,and bioinformatics analysis of INH alpha protein was performed.(2)The xinjiang Bactrian camel was immunized with the purified INH alpha recombinant protein to prepare the camel anti-INH alpha polyclonal antibody and purify it.The antibody titer was detected by indirect ELISA method,and the specificity of the binding of the antibody to the target protein was verified by Western blotting.(3)The 45 Kazakh sheep were randomly divided into three groups:Group A(INH polyclonal antibody immune group),Group B(INH recombinant protein immune group),and group C(control group),the contents of reproductive hormones in sheep blood of the three groups were detected by ELISA kit,and the biochemical indexes of sheep blood were detected.Finally,the data were analyzed by SPSS software.Results:(1)In IPTG of 37℃ and final concentration of 0.1mmol/L,INH alpha protein was largely expressed as inclusion body for 4h after induction.After purification and enterokinase digestion,INH alpha protein was highly purified and immunogenic.Bioinformatics analysis,according to the results of INH alpha is contain a signal peptide,but without an unstable across the membrane structure is a fat soluble protein,in the secondary structure,INH alpha contains the alpha helix is occupied 16.11%,beta Angle is occupied 3.06%,extending chain 16.94%,no rules curly accounted for 63.89%,INH alpha has three kinds of tertiary structure model of highsimilarity,among them,the highest and reliable model 2 C is worth points.(2)After 70 days of immunization,the antibody titer in bactrian camel reached 1:52000.The antibody can specifically bind to INH alpha protein.(3)Before INH alpha antigen immunization,FSH,LH,E2,INH and P4 levels were in equilibrium among the three groups,with no significant difference between the groups(P>0.05).After immunization,FSH content in group A was significantly higher than that in group C(P<0.05),while FSH content in group B was higher than that in group C,but the difference was not significant(P>0.05).The LH content in group A and group B was lower than that in group C,but the difference was not significant(P>0.05).The content of P4 in group A and group B was higher than that in group C,but the difference was not significant(P>0.05).The E2 content of group A was significantly higher than that of group C(P<0.05),and the E2 content of group B was higher than that of group C,but the difference was not significant(P>0.05).The INH content in group A was significantly lower than that in group C(P<0.05),while the INH content in group B was lower than that in group C,but the difference was not significant(P>0.05).Blood biochemical tests of sheep showed that neither the INH alpha protein nor the INH alpha polyclonal antibodies had adverse effects on liver,heart or kidney function of sheep.The antigens were safe.Conclusion:(1)The immunogenic INH alpha protein was successfully induced and purified in the form of inclusion bodies.(2)INH alpha immunity produced a large number of antibodies in the camels.(3)Both INH alpha protein active and passive immunity had certain effects on reproductive hormones of Kazakh sheep,but compared with active immunity,INH alpha passive immunity had better effects.Moreover,the two antigens had no significant effects on organ function of Kazakh sheep and had no toxic effects.