非洲猪瘟病毒截短p54蛋白的原核表达与纯化

Prokaryotic Expression and Purification of p54 Truncated Protein of African Swine Fever Virus

旨在高效表达可溶性的非洲猪瘟病毒(ASFV)p54蛋白。去除可能影响p54蛋白可溶性表达的前50个氨基酸(aa),根据GenBank收录的ASFV p54基因序列设计引物,从第51个aa对应的基因序列起用PCR扩增截短的p54基因,并用分子克隆方法插入到带有TF及His标签的冷休克表达载体pCold TF上,构建pCold TF-p54重组质粒。经菌液PCR及测序验证后将重组质粒转化到BL21感受态细胞中,IPTG诱导表达p54重组截短蛋白,对IPTG诱导浓度、诱导时间进行优化,并对纯化重组蛋白时所用洗脱液的咪唑浓度进行探索。Western blot鉴定p54重组截短蛋白的反应原性,并探究TF标签对于该蛋白反应原性的影响。结果表明,成功构建了pCold TF-p54重组质粒,经终浓度为1 mmol/L的IPTG于16℃诱导9 h时,p54重组截短蛋白的表达量最高,分子质量约为76 ku。经50 mmol/L咪唑洗脱液洗脱可得到较纯的p54重组截短蛋白,经人鼻病毒(Human rhinovirus,HRV)3C Protease切除TF及His标签后,再次纯化可得到无标签且高纯度的p54截短蛋白。Western blot结果显示无标签的p54截短蛋白及有标签的p54重组截短蛋白均可被ASFV p54单克隆抗体特异性识别,均具有反应原性,且p54重组截短蛋白还可被ASFV阳性血清特异性识别。研究证实了原核表达的p54重组截短蛋白兼具可溶性、良好的反应原性,且重组蛋白上的TF标签不影响抗体对p54蛋白的识别,因此可用于后续ASFV抗体检测方法的研发。

In order to efficiently express soluble African swine fever virus(ASFV)p54 protein,the first 50 amino acids(aa)that may affect the soluble expression of p54 protein were removed,the primers were designed according to the ASFV p54 gene sequence in GenBank,the truncated p54 gene was amplified by PCR from the gene sequence corresponding to the 51st aa,inserted it into the Cold-Shock expression vector pCold TF with TF and His tags by molecular cloning method,and constructed the pCold TF-p54 recombinant plasmid.The recombinant plasmid was transformed into E.coli BL21 competent cells after the verification of bacterial PCR and sequencing.The expression of p54 recombinant truncated protein was induced by IPTG.The induction concentration and induction time of IPTG were optimized,and the concentration of imidazole in the eluent used to purify the recombinant protein was explored.The antigen reactivity of p54 recombinant truncated protein was identified by Western blot,and the effect of TF label on the antigen reactivity of p54 recombinant truncated protein was explored.The results showed that the recombinant plasmid pCold TF-p54 was successfully constructed.When induced by IPTG with a final concentration of 1 mmol/L at 16℃for 9 hours,the expression level of p54 recombinant truncated protein was the highest,and the molecular weight was about 76 ku.The purified p54 truncated protein can be obtained by eluting with 50 mmol/L imidazole eluent.After the TF and His labels were removed by HRV 3C protease,the unlabeled p54 truncated protein with high purity can be obtained by purification again.Western blot results showed that both unlabeled p54 truncated protein and labeled p54 recombinant truncated protein could be specifically recognized by ASFV p54 monoclonal antibody,and both had antigen reactivity,and p54 recombinant truncated protein could also be specifically recognized by ASFV positive serum.This study confirmed that the prokaryotic expression of p54 recombinant truncated protein has both solubility and good antigen reactivity,and the TF label on the recombinant protein does not affect the recognition of p54 protein by the antibody,so it can be used for the development of subsequent ASFV antibody detection methods.