Cloning, Expression And Antigenicity Identification Of Newborn Larvae (NBL) Stage-specific Gene In Trichinella Spiralis

Trichinellosis is a kind of zoonosis caused by Trichinella spiralis (T.s). It not only results in serious threat to human health but also great economic damage to livestock farming with its morbidity and mortality being high in parasitic zoonosis. So T.s is an important item in food sanitary examination in China. Because the species and strain exist wide variation in biology epidemiology and pathology in different hosts and geographic regions, clinical diagnosis of T.s is very difficult. With the development in molecular biological technology, there is a new breakthrough in T.s research.The cDNA library of Trichinella spiralis (T.s) newborn larvae (NBL) was screened by NBL stage-specific probe T668 labeled with DIG. A positive cDNA clone of 1609bp in length with an open reading frame (ORF) of 1395bp was obtained. The cDNA encodes a polypeptide of 465 amino acid residues with the predicted molecular weigh of 50.1kDa and IP of 9.7. Bio-soft analysis showed 31-64 amino acid residues of the peptide were signal peptide sequence, 31-64 amino acid residues of N terminal were mainly hydrophobicity and hydrophilic regions mainly situated C terminal. Blastn homology analysis indicated that the cDNA, named T668 was a novel and had no obvious homology to any other known gene sequence. It was registered in GenBank and access number was AF 331160.1. Blastp homology analysis indicated that the homology to serine protease was 38%. Meanwhile, a cDNA fragment of T668 was obtained and named G10-7. G10-7 and T668 without signal peptide sequence were obtained from pBK-CMV-G70-7 and pBK-CMV-T668 by PCR and subcloned into prokaryotic expression vector pET28 and the recombinant expression plasmids (pETG10-7, pETT668) were successfully constructed. The expression plasmids were transformed into E.coli strain DE3, and induced by ImM IPTG Two recombinant fusion proteins of 34kDa and 49kDa were identified by SDS-PAGE respectively. The amount of the expressed proteins increasedwith time extending and reached 61.7 % and 34.6 % of the total bacterial protein after inducing 4 hours and 5 hours respectively. Western blotting showed the two recombinant proteins were recognized by positive sera from swine infected with T.s.In order to study the specificity, sensibility of recombinant proteins in diagnosis of Trichinellosis and immunoprotective to mouse, G10-7 and T668 recombinant protein as diagnostic antigen and goat-anti-rabbit IgG, goat-anti-pig IgG, goat-anti-human IgG labeled with HRP as secondary antibody, indirect ELISA of detecting T.s antibody was established, while excretory-secretory (ES) antigen as the control. The results showed these two kinds of recombinant proteins had no difference from ES antigen in diagnosis. For determination the immunogenicity of the recombinant proteins, the mice were immunized for 3 times at 10 days interval. Two weeks after the final immunization, the animals were challenged with 200 T.s infective muscle larvae (ML) per mice. ML loading was detected with digest method 5 weeks after challenge infection, the IgG titer in the serum 1-5 weeks after challenge infection was detected by ELISA. The results showed recombinant protein protected effectively against T.s challenge in immunized groups, the reduction rate of ML (T668: 67.0%, G10-7: 63.3 %) and IgG titer were significantly higher than that in the adjuvant and control groups. It showed that T668 and G10-7 recombinant proteins could induce protective immunity and specific humoral immunity.On the base of prokaryotic expression, the primers were designed according to T668 and G10-7 gene sequences and the fragments of G10-7 and T668 including signal peptide were amplified with pBK-CMV-7tf6S and pBK-CMV-G70-7 as templates, then they were cloned into eukaryotic expression vector pCR3.1. Expression plasmids pCRG10-7 and pCRT668 were successfully constructed and transfected into COS-7 cell. The expression protein was detected by ELISA in cell lysate and culture supernatant at different culture time. The results showed T668 and G10-7 were expressed...