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First of all, according to GenBank accession SLA-DRA and SLA-DRB gene sequence-specific primers, both ends of the primers, respectively, plus the BamH I platinum and Xho I restriction sites and to protect the base. By reverse transcription-polymerase chain reaction (PT-PCR) technology from by Landrace pig intestines lymph node tissue total RNA was amplified by two specific gene fragments. After the separation and purification of PCR products connected to the pMD18-T vector, positive recombinant clones screened by the bacilli PCR and double digestion reaction and sequenced. The results showed that the test was successfully cloned SLA-DRA and SLA-DRB gene, respectively, 759 and 801 nucleotides, encoding 252 and 266 amino acids. Bioinformatics prediction of the structure and function of the SLA-DRA and SLA-DRB found that the SLA-DRA amino acid sequence has two potential N-glycosylation sites, Asn141, Asn244 glycosylation, SLA-DRB 1 potential N-glycosylation sites in the amino acid sequence, Asn48 is glycosylated. SLA-DRA There are four major hydrophobic region, and a strong hydrophilic SLA-DRB performance. SLA-DRA amino acid sequence has 12 potential phosphorylation sites: Ser38, Ser42, Ser 116, Ser156, Ser179, Thr64 and Thr97, Thr207, Thr216, Thr246, Tyr36 and Tyr184, SLA-DRB amino acid sequence 13 potential phosphorylation sites: Ser71, Ser92 Ser196, Ser208, Ser221, Ser223, Thr32 and Thr50 and Thr80, Thr129, Thr186, Thr210 and Tyr107. In this study, SLA-DRA, SLA-DRB gene directional cloning into the prokaryotic expression vector pET32a () to construct a recombinant fusion pET32a-DRA expression of elemental particles, the pET32a-DRB transformed E. coli BL21 (DE3) induced by IPTG optimize the expression conditions, and preliminary purification of recombinant fusion protein, the results showed that: SLA-DRA and SLA-DRB gene in the recombinant fusion expression plasmid pET32a () in the direction of insertion and correct reading frame; SLA-DRA and SLA-DRB gene fusion expression in E. coli BL21 (DE3), the expression level accounted for 19.88%, 17.26% of the total bacterial protein expression conditions were optimized, and the expression level reached 26.22%, 21.98%: induction 2h after the pET32a-DRA fusion protein reached peak induction after 3h the pET32a-DRB fusion protein expression levels peak. Through the stationary phase particle affinity chromatography (IMAC) purification of the recombinant fusion protein, wherein the imidazole concentration of 300mmol / L of Elution buffer the effect of purification are preferably purified recombinant fusion protein specificity.
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