Expression Of Trichinella Spiralis Skin Collagen Rol-6 In Caenorhabditis Elegans

The epidermis was composed of T.spiralis cuticle collagen can not only maintain body shape and movement, is also able to resist the host defense system and protect the parasite from external injury. With the study of parasite’s cuticle collagen such as potato hairworm、globus pallidus cyst nematodes、schistosome, proved the kind of protein is the most important antigen and can cause host immune response. The cuticle collagen of T.spiralis synthesis is fast after ecdysis, although structure and composition are different in every stage, immune response is same as other nematodes that can cause strong immune responses in the host. In recent years, research of skin collagen focuses on potato hairworm and schistosomiasis, the skin collagen rol-6 of T.spiralis is few, Thus using C.elegans to express rol-6 protein can lay a foundation for vaccine to screen potential antigens of T.spiralis.In this study, total RNA was extracted by T.spiralis and reversed transcription to c DNA as template, designed primers according to the sequence of rol-6 gene to amplify, then cloned the target fragment to pMD19-T and transformed into Tans5α. The results detected by PCR and Bam H1/Sal I digested identified. Sequencing results showed that the cloned sequence included1023 nucleotides which can encode 340 amino acids. Using bioinformatics software to analyze the sequence structure, the results show that rol-6 not only has the structure of the skin collagen Gly-X-Y, but also has the potential of epitope. The rol-6 encoding sequence was cloned into pGEX-6p-1 expression vector to construct recombinant plasmid pGEX-rol and induced to express.The results showed that the recombinant protein has a molecular weight of about 61 KD, which supports GST tag in the protein is about 26 KD, the rol-6 protein is about 35 KD, consistent with the expected results. Using the recombinant protein immunized mice successfully obtained rat anti rol-6 serum.For further expressing rol-6, using PCR method from C.elegans genomic DNA amplification sqt-1 5’flanking sequences( 5’-FSsqt-1) as a promoter, 5’-FSsqt-1 、 rol-6 coding sequence was cloned into pPD95.77 which contains green fluorescent protein(GFP)to construct recombinant plasmid pPD-FSsqt-rol-GFP, then injected into the C.elegans gonad and cultured. The results showed that 5’-FSsqt-1 was able to activate the transient expression of rol-6 and GFP fusion protein in C.elegans.T.spiralis and C.elegans are nematodes, with a genetic relationship, the alternative splicing,glycosylation and folding conformation will closer to natural antigen. Thus, the antigen activity is enhanced, and the expression process is simple and convenient, which provides convenience forthe development of T.spiralis vaccine.