Cloning, Expression And Diagnostic Properties Of Antigenic Genes In Trichinella Spiralis At Different Developmental Stages

Trichinellosis is a serious foodborne parasitic zoonoses with worldwide.Although it has been discovered for more than 200 years,there are still a large number of cases and outbreaks in many parts of the world each year.Since the clinical symptoms of trichinellosis are not specific,it is not easy to be diagnosed,especially early diagnosis.So far,there is no quick,accurate and convenient diagnostic method.The aim of this study is to find a highly sensitive,specific,and large-scale preparated antigen.Objective:This study is based on the previous research foundation of trichinella spiralis,which four strong reactive antigen genes have been found in different development periods.Then they were cloned and expressed in vitro and made to gene recombinant proteins.The antiserum of mice infected with trichinella and trichinellosis human serum were used to evaluate gene recombinant proteins' diagnosis effect so that obtain the candidate gene recombinant antigen which can be used for the diagnosis of trichinellosis.Methods:According NCBI-Gene,the intestinal muscle larval stage(inML6h)class cysteine protease inhibitors-like(CPIL)gene Wn10,adult Ad3 serine protease(SP1)gene Zh68,newborn larvae stage(NBL)specific serine protease(SP2)gene T668,muscle larva(ML)serine protease inhibitors(Serpin)gene Wm5 have been named and their gene sequences have been discovered.Bioinformatics methods can be used to predict four genes coding proteins' structures and functions.By analyzing gene sequences,primers were designed,the appropriate carrier was chosen.Then four genes were cloned and expressed in vitro,thus four recombinant proteins were obtained,respectively named P1,P2,P3 and P4.Furthermore,the change of antibody in mice serum with different infected doses and time detected by four recombinant antigens were observed by ELISA.At the same time,serum of trichinellosis patients were used to compare the diagnostic effect of four ES proteins and four gene recombinant proteins and commercial kits by ELISA.All data obtained were showed and analyzed by SPSS 22.0 and Excel 2016.Results:According to the predicted results of bioinformatics,the proteins encoded by the 4 genes are hydrophilic proteins,and SP2 has a transmembrane region.CPIL,SP1 and SP2 contain signal peptides.Four proteins all have numerous epitopes of antigen,and the dominant epitopes are selected.Four highly purified recombinant proteins were successfully obtained by using E.coli expression system and protein purification technology.Further,their diagnostic characteristics were identified using ELISA method:1)the results of mice serum showed that,at the IgM level,the detection levels of 4 recombinant proteins were lower or negative.At IgG level,the first time detected positive of P1 at low dose group and high dose group is 10dpi and 8 dpi,antibody level of 11dpi-45dpi maintains a high level,and early infection antibody level of low dose group was obviously higher than that of high dose group.The earliest day can be detected the positive by P2,P3,P4 is 10 dpi,15dpi,16 dpi.2)The results of human serum showed that,at IgM antibody level,all four ES antigens and P2 and P3 can detect IgM antibody of all serum of patients with trichinellosis;at IgG antibody level,all four ES antigens and P4 and commercial kit can diagnose IgG antibody of all patients with trichinellosis.Conclusion:Four recombinant proteins have good diagnostic features when IgG antibody of infected mice serum are detected.Among of them,P1 can be used as a candidate antigen for early diagnosis,P4 can be used for medium-term or later period.At the same time,we found that antibody levels are significant different in four infected doses when detected by P1,which low dose group was obviously higher than high dose group,we speculated that it may be involved in the early infection of host immune suppression function,its biological function and application prospects still need further research.When patient sera were used to identify the diagnostic features of four gene recombinant proteins,the results show that the P2 and P3 can be used for the detection of trichinellosis IgM antibodies,P4 can be used as antigen for IgG antibody.The diagnostic characteristics and application values of P1 and P4 are needed to be further improved and further studied.