结核分枝杆菌分泌蛋白Rv0674的特异性结合肽的筛选

Screening for Specifically Binding Peptide of Rv0674 from Mycobacterium Tuberculosis

目的制备结核分枝杆菌(Mycobacterium tuberculosis,MTB)分泌蛋白Rv0674,筛选与Rv0674蛋白特异性结合的肽分子。方法用PCR从MTB(H37Rv)基因组中扩增出Rv0674基因。克隆到pEASY-E1表达载体上,将测序验证正确的重组表达质粒转化入E.coli BL21中,用IPTG诱导蛋白表达,Ni2+亲和层析法纯化目的蛋白。通过SDS-PAGE和Western blot鉴定后,将纯化后的Rv0674蛋白包被到96孔板上,再利用噬菌体展示技术,经过连续4轮特异性亲和筛选,对第4轮产物随机挑取单独的噬菌斑,提取基因组并测序,用SnapGene 4.1.4比对并翻译多肽的基因序列。结果构建了Rv0674序列正确的重组表达质粒,获得分子量约为26.5kD的可溶性Rv0674蛋白。从第4轮的洗脱物中,随机挑选40个噬菌斑。测序结果可翻译成10种多肽分子,其中重复19次的多肽序列为TSTMMMHMNL。结论通过噬菌体展示技术筛选到TSTMMMHMNL多肽序列,对Rv0674蛋白的亲和力和特异性最好。

Objective To prepare the secretory protein Rv0674 of Mycobacterium tuberculosis and screen the peptide molecules specifically binding to Rv0674 protein.Methods PCR was used to amplify Rv0674 gene from MTB H37Rv genome.Rv0674 was cloned into pEASY-E1 expression vector,and the gene was sequenced.The plasmid with Rv0674 gene was transformed into E.coli BL21.The expression of Rv0674 protein was induced by 1.0 mM IPTG at 28℃for 8h.The Ni2+affinity chromatography was used to purify Rv0674 and identified by SDS-PAGE and Western blot.A phage-displayed random seven-peptide library was used to screen Rv0674 purified protein.The purified Rv0674 protein was coated into a 96-well plate at a concentration of 100μg·mL-1.The phages,which tightly bound to Rv0674,were obtained after four rounds of“adsorption-panning-elution-amplification”.Forty plaques were selected to extract genome and sequence by extension primer-96gIII.The phage sequencing results were aligned and translated by SnapGene 4.1.4 software.Results A recombinant expression plasmid with the correct sequence of MTB Rv0674 was constructed.The soluble Rv0674 protein,approximate 26.5 kDa,was expressed and purified.After four rounds of phage display,40 plaques were randomly selected to sequence.The sequencing results could be translated into 10 polypeptide molecules,wherein 19 plaques with the same peptide(TSTMMMHMNL).Conclusion The polypeptide TSTMMMHMNL,which has the best affinity and specificity for Rv0674,was screened by phage display technology.