Cloning And Fusion Expression In Escherichia Coli Of Porcine MyD88 Gene

Specific primers for MyD88 were designed through GenBank database, EcoR I and Not I restriction enzyme cut sites and protective bases were added at the ends of the primer.Total RNA was extracted from mesenteric lymph node(MLN) of Landrace by TRIzol reagent,a specific ragment was amplified by reverse transcription-polymerase chain reaction(RT-PC R).Subsequently,the fragment was ligated to pMD18-T vector after purified, then recombinant sequence was identified by PCR and double digests method. The results suggest that the fragment contained 882 base pairs and the deduction protein contained 293 amino acids after sequencing. Then the cloned amino acid sequence was analyzed using the online sofeware. The results suggest that the structure and function of MyD88 gene were predicted by bioinformatics methods. The deduced amino acid sequence of MyD88 gene possessed three typical functional domain including an N-terminal death domain, as well as intermediate and C-terminal TIR domains.Besides,Three potential O-glycosylation sites,eighteen potential phosphory sites and Three main hydrophilic in MyD88 amino acid sequence were found,but no N-glycosylation sites and transmembrane domain .The main secondary structure of protein was composed of randon coil.The alignment of amino acid sequences of MyD88 gene showed that the sequence was highly conserved among human, cattle, rat and mouse,the TIR domain of MyD88 amino acid sequence was 98.53% identical to that of the human's.Which indicated that MyD88 of porcine was more similar to MyD88 of human in amino acid sequence.The MyD88 gene was inserted into prokaryotic espression vector pET32a (+) and we constructed recombinant fusion expression plasmid pET32a-MyD88. Then pET32a- MyD88 was transformed into E.coli BL21 (DE3) and induced by IPTG. The expressed condition was optimized and the recombinant fusion protein was purified. The results showed that the insert direction and reading frame of MyD88 gene were successful. The pET32a-MyD88 gene had already expressed in E.coli BL21 (DE3). The fusion protein expressed was about 53.6KDa and mainly exised as inclusion bodies. The expression capacity was 19.38%, and it was increased to 23.22% by optimizing expressed conditions.The expression capacity of pET32a-MyD88 reached peak value at 2h after induction.The recombinant fusion protein was purified by immobilized metalion affinity chromatography (IMAC) methods.The purification effect of Elution buffer which contained 300mmol/L imidazole got the best result.The recombinant bands of fusion protein were specific,so. The MyD88 gene of porcine was expressed successfully by E.coli expression system, which provided the genetic character of this gene in further study.