Gene Synthesis, Expression And Function Analysis Of Egg Protein IPSE Of Schistosome Mansoni

Schistosomiasis is an important endemic disease that harms to millions of people. The main reason that schistosome can exist in human blood vessels for a long period if unchallenged by drug treatment,is because excretory/secretory antigen can bias the immune reaction,from Th1 response--protecting host from infection,towards Th2 response--profiting growth and development of parasite.Excretory/secretory antigens deriving from adult worm or egg exposed in host immunity system,which is the direct envidence of alive schistosome infection.The detection of these molecules or antibodies against them is very important for diagnosis of schistosomiasis.Study on these molecules also helps to clear the immune mechanism of schistosome infection, which not only promoting development of high effective vaccine and adjuvant,but also improving prevention measures for autoimmune disorder.Earlier study shows IL-4-inducing principle of S.mansoni eggs(IPSE) is a secretory glycoprotein,which is enriched in the subshell area of schistosome eggs. There is a high sensitivity and specificity in detecting specified antibody in schistosomiasis serum with IPSE,and it can be use for accessing effect of chemotherapy in some certain.At the same time,this molecule is an effective regulator leading to the expression of IL-4,which skewing immune response towards Th2.There is no report on IPSE of Schistosomo.japonium,so,for the sake of isolation of IPSE similar gene from S.japonicum cDNA libraries in the future,we synthesized the IPSE gene according to the IPSE DNA sequence of S.mansoni,and expressed it's recombinant protein with nature immunological activity.Hopefully,the study on IPSE gene can serve for the development of new method of schistosomiasis diagnosis and intervention measures.This study includes three parts,as follows:1.Gene synthesis and identification of IL-4-indueing principle of S.mansoni eggs(IPSE)Fourteen oligonucleotides fragments(each of 50 bp) with complementation ends has been designed according to the DNA sequence of mature peptide of IPSE,were synthesized by chemical method.With the fourteen oligonucleotides fragments as primer,the whole DNA fragment of IPSE(about 417 bp) was synthesized by carrying out overlapping polymerase chain reaction(overlapping PCR).Then,the gene fragment of IPSE was inserted into pGEM-T vector and sequenced.Sequencing data showed that the DNA sequence of synthesized IPSE is completely identical to the natural one,which means the gene synthesis of IPSE was successful in this study.2.Construction of prokaryotic expression plasmid of IPSE and protein expressionFor construct a recombinant expression plasmid IPSE/pET32 a(+),the gene of IPSE was subcloned into expression vector pET32a(+) at the downstream of thiredoxin(Trx).Then,the recombinant expression plasmid was transformed into host bacteria E.coli BL21(DE3).The single colony bacteria containing recombinant expression plasmid was induced by IPTG and expressed a 35.7 kDa recombinant fusion protein which correspond to predicted molecular weight of fusion protein.3.Purification,refold and function analysis of Trx-IPSE fusion proteinLarge scale of fusion protein were prepared and purified with Ni affinity chromatography gel under denaturing conditions.A small mount of soluble Trx-IPSE were got by dialyzing the fusion protein in a large volume of PBS.Western blotting result showed that the Trx-IPSE protein could be recognized by anti-IPSE IgG antibody in the pool serum of schistosomiasis,no matter under reducing or non reducing conditions.It also showed with goat anti-human IgE antibody as second antibody,Trx-IPSE fusion protein could be recognized non-specifically by IgE in the sera of healthy persons,it means fusion protein has capacity of binding IgE antibody.In this study,we successfully synthesized the IPSE gene of S.masoni and got fusion protein with bioactivity similar to nature protein.It's made a better foundation for discovering similar gene of S.japonicum by isolating IPSE cDNA from S. japonicum egg cDNA libraries with antibody against IPSE in future,and it may promote development of high effective diagnostic methods,vaccine,adjuvant and prevention measure for autoimmune disorder.