Identification And Functional Analysis Of Serpin-1like Gene From Oak Silkworm Antheraea Pernyi

Antheraea pernyi be founded in China shandong province at the earliest and begin to domesticated.It is important wild spun silk insect of China.Serine protease inhibitors(serpin)can restrain with serine protease adjusting many life activities in organisms to achieve dynamic balance in the course of insect innate immune.Serpin play an important role in innate immunity of insects.We mainly studied the Antheraea pernyi serpin-1like gene expression patterns and the function in the insect innate immune.Research results are as follows:In this study,through PCR method to clone and get serpin-1like gene which the open reading frame(ORF)was 837 bp.It contains a conservative serpin structure domain,The evolutionary tree analysis showed that it was closest relative with Antheraea mylitta.Homology analysis showed that it was 82.1% homology with Antheraea mylitta.Through builded pET-28a-Ap-serpin-1like vector,by different concentrations of IPTG induce with pET-28a-Ap-serpin-1like protein.The result was that Successfully expressed Antheraea mylitta serpin–1like protein.After a lot of induced use the Ni-NTA column purified the restructuring pET-28a-Ap-serpin-1like proteins get the high purity pET-28a-Ap-serpin-1like protein.The protein concentration was about 0.5 mg/ml.Use the pET-28a-Ap-serpin-1like protein was employed as antigen in batches to immunize a New Zealand white rabbit,taking blood to get the Antheraea mylitta serpin-1like polyclonal antibody serum and detect the antibody titer.The result of antibody titer was about 1:128000.qRT-PCR and Western blotting results showed that Ap-serpin-1like was widely expressed in Antheraea pernyi six tissue in larvae at the third day of the fifth instar stage.The results show that the Ap-serpin-1like belongs to constitutive express in Antheraea pernyi.In Antheraea pernyi fat body the expression higher than other tissue,but in Antheraea pernyi hemolymph the expression was smaller compared with other tissue.At the same time,qRT-PCR analysis of Ap-serpin-1like expression levels at different developmental stages of Antheraea pernyi.The result indicated that Ap-serpin-1like expression was greatest at the pupal stage and lowest at the 4th larval stage.The recorded was insignificant variations at other developmental stages.Then we study when the four kinds of pathogenic microorganism influence five age three days of Antheraea pernyi,the changes of the Antheraea pernyi immune function.In the three tissue(fat body,hemolymph and midgut)Ap-serpin-1like transcript expression can be obviously raise or decline when the pathogenic microorganism injected.It was found that: In the fat body,except treated with B.bassiana,Ap-serpin-1like expression down regulate from 24 h to 48 h.Other three pathogenic microorganism stimulate,Ap-serpin-1like expression all upregulate,but each time the increase degree was different.In the hemolymph,inject with B.bassiana and M.luteus,Ap-serpin-1like mRNA expression was highest at 24 h.When treat the E.coli was highest at 1.5 h.In the midgut,inject with B.bassiana Ap-serpin-1like mRNA expression was higher at 48 h than other time.The expression of Ap-serpin-1like was highest at 24 h when treat with NPV.When M.luteus induced,Ap-serpin-1like expression highest at 1.5 h and the E.coli was little effect to Ap-serpin-1like mRNA expression.In conclusion,Ap-serpin-1 like gene in Antheraea pernyi may have important role and function in congenital immune response.Our research can explain the serine protease inhibitors function in the process of Antheraea pernyi physiological pathology and also have important referential meaning in the study of serine protease inhibitors functions in other animals,plants and microorganisms different life activities.