Cloning, Expression And Identification Of Aquaporin Gene Of Trichinella Spiralis

Trichinellosis is a food-borne parasitic zoonosis with global distribution which is caused by Trichinella spp.Trichinella spp. can infect more than 150 species of animals including humans, and can lead to serious diseases or even death. Meanwhile, trichinellosis can cause enormous economic losses to development of livestock husbandry. Aquaporins are water channels that widely existed in living organisms, belonging to major intrinsic protein family, which are potential drug targets and candidate vaccine antigen. In this study, the specific primers for T. spiralis aquaporin gene(TsAQP) were designed based on EST sequences derived from GenBank database and RT-PCR was performed to turn the total RNA of T. spiralis muscle larvae into cDNA by Universal primer PR1, then the specific primers were used to amplify the open reading frame sequence of TsAQP gene from cDNA. The bioinformatic analysis indicated that the TsAQP gene contains 6 exons and 5 introns. The full length of the open reading frame was made of 867 base pair and encodes 288 amino acids. The theoretical molecular weight of TsAQP was 31.01 kDa and isoelectic point(pI) was 8.47. TsAQP contains 5 loops(A-E) with conserved NPA(Asn-Pro-Ala) motif in B and E loop, respectively. As the composition of secondary structure, α-helix accounts for 34.38%, β-turn accounts for 9.38%, the rest were extended strand and random coil. The TsAQP has a 45.0% sequence identity to human HsAQP9 and the phylogenetic analysis demonstrated that TsAQP belongs to the aquaglyceroporins subfamily. The 121-151, 78-97(B loop), 210-238(E loop) and 262-288 amino acids were cloned and ligated into pGEX4T-1 prokaryote expression vector, and then the recombinant expression vectors were induced to express after transformed into E.coli BL21 by IPTG. The molecular weights of these recombinant proteins were 29.3, 28.1, 29.3, 29.0 kDa, recpectively. The non-transmembrane region of TsAQP gene was artificially synthesized and ligated into pET30 a vector. The molecular weight of recombinant protein was 25.2 kDa and mainly existed in the form of inclusion body. The recombinant protein could be recognized by infected T. spiralis pig sera. Quantitative Real Time-PCR results indicated that TsAQP gene could express through the entire life cycle, with a higher transcription level in the new borne larvae. Immunohistochemistry analysis showed that TsAQP was mainly located in the tegument of T. spiralis muscle larvae. BALB/c mice immunized with mixed proteins(GST-121/151, B, E, 262/288) and TsAQPnt could generate high level antibodies. And after challenged with T. spiralis muscle larvae, the antibody level showed a slightly decreased tendency. The results showed that the muscle larvae numbers were reduced 20.6% and 14.7% after BALB/c mice immunized with mixed proteins(GST-121/151, B, E, 262/288) and TsAQPnt compared to that in controls, which indicated that the recombinant proteins would be potential to induce protective immunity in BALB/c mice against the infection of T. spiralis muscle larvae. The present study will lay the foundation for the further study of the biological function of T. spiralis aquaporin.