人内毒素结合肽基因的突变及突变体蛋白表达

目的分析亲代人内毒素结合肽一级结构,选择可能影响其生物学活性的氨基酸对应碱基位点进行突变,克隆基因突变体mEBP基因并研究其蛋白表达。方法以亲代EBP基因为基础,结合DNASIS软件理化性质分析确定突变碱基,应用PCR定点诱变技术进行第5、18位谷氨酰胺→赖氨酸的定点突变。并将其克隆至原核融合表达载体pinpointXa-3,转化大肠杆菌BL21(DE3)pLysS进行表达,经Western blot鉴定。结果突变EBP基因13及52位核苷酸由C突变为A,构建的阳性重组子经酶切及测序鉴定证实与设计序列完全一致,经IPTG诱导在大肠杆菌BL21(DE3)pLysS表达生物素化的融合蛋白,Western blot鉴定显示能与抗生物素单克隆抗体结合。结论已成功获得EBP基因突变体,并在大肠杆菌以融合蛋白的形式进行表达。

Mutation and Expression of Human Endotoxin Binding Peptide Gene

Objective To analyze the primary structure of parental human endotoxin binding peptide(EBP),induce mutation at base sites corresponding to the amino acids which might influence the biological activity of EBP,clone the mutant of EBP(mEBP) and study its expression.Methods mEBP gene was predicatively analyzed by DNASIS software,and the mutations(Gln→Lys) at sites 5 and 18 were induced by PCR site-directed mutagenesis.The mEBP gene was cloned into prokaryotic vector pinpoint Xa-3 and transformed to E.coli BL21(DE3) pLysS for fusion expression.The expressed product was identified by Western blot.Results The nucleotides at sites 13 and 52 of mEBP gene were mutated from C to A.The constructed recombinant showed identical sequence to that designed,as proved by digestion with restriction endonuclease and sequencing.The biotinylated fusion protein was expressed in E.coli BL21(DE3) pLysS under induction of IPTG.Western blot showed the binding capacity of expressed product to McAb against biotin.Conclusion The mutant of EBP gene was obtained and successfully expressed in E.coli in a form of fusion protein.