Cloning And Fusion Expression In Escherichia Coli Of SLA-DRA,SLA-DRB

Firstly,specific primers for SLA-DRA and SLA-DRB were designed by GenBank, BamHâ… and Xhoâ… restriction enzyme cut sites and protective bases were added at the ends of the primer respectively.Total RNA was prepared from mesenteric lymph node(MLN) of Landrace according to the instructions of the TRIzol reagent,two gene fragments were amplified by reverse transcription-polymerase chain reaction(RT-PCR).Subsequently,gene fragments were ligated to pMD18-T vector after purified,the recombinants were sequence and analysed aider they were identified by PCR and double digests.The results suggest that the obtain gene fragments contain 759 and 801 base pairs and translate into proteins containing 252 and 266 amino acids,respectively.The structure and function of SLA-DRA and SLA-DRB were predicted by bioinformatics methods.Two potential N-glycosylation sites in SLA-DRA amino acid sequence was found, Asn141 and Asn244 could be glycosylated,and one potential N-glycosylation sites in SLA-DRB amino acid sequence was found,Asn48 could be glycosylated.Four main hydrophobicity region of SLA-DRA was found,and SLA-DRA had hydrophilic property.Twelve and thirteen potential phosphory sites in SLA-DRA and SLA-DRB respectively.The SLA-DRA and SLA-DRB was inserted into prokaryotic espression vector pET32a(+).Thus we constructed recombinant fusion expression plasmid pET32a-DRA and pET32a-DRB.Then pET32a-DRA and pET32a-DRB were transformed into E.coli BL(DE3) and expressed with induction of IPTG.The expressed condition was optimized and the recombinant fusion protein was purificated.The results showed:Both the insert direction and reading frame of pET32a-DRA gene and pET32a-DRB gene at the cloning junction were correct;both pET32a-DRA gene and pET32a-DRB gene had already expressed in E.coli BL(DE3).The expression capacity were 19.88%and 17.26%respectively,and they could be increased to 26.22%and 21.98%by optimizing expressed conditions.The expression capacity of pET32a-DRA reached peak value in 2h after induction,and the expression capacity of pET32a-DRB reached peak value in 3h after induction.The recombinant fusion protein was purificated by immobilized metal ion affinity chromatography(IMAC) methods.The purification effect of Elution buffer which contained 300mmol/L imidazole are all better.The recombinant bands of fusion protein are special.