伴放线放线杆菌菌毛融合蛋白基因的克隆

Cloning and Sequence Analysis of Recombinant Fusion Gene of Escherichia Coli Heat-liable Enterotoxin B Subunit and Actinobacillus Actinomycetemcomitans Fimbria Associative Protein

目的 探讨克隆伴放线放线杆菌菌毛融合蛋白基因ltb_fap的方法。方法 采用PCR方法扩增伴放线放 线杆菌菌毛相关蛋白(Fap)和大肠杆菌不耐热性肠毒素B亚单位(Ltb)的融合蛋白基因ltb_fap,NcoⅠ EcoRⅠ双酶切 载体pET28a(+)及ltb_fap基因,连接形成重组质粒pETltb_fap,转化至大肠杆菌DH5α,扩增后提取质粒pETltb_fap进 行PCR鉴定、酶切鉴定和序列分析。结果 本实验扩增的ltb基因约为303bp,fap基因约为228bp,融合基因约为 531bp。重组质粒含外源基因片段约为531bp,酶切鉴定可得到一条约531bp带,DNA测序结果表明与GenBank中 的fap基因序列有97.4%的同源性。结论 成功克隆出融合蛋白基因ltb_fap,为进一步进行蛋白表达、制备抗体奠 定基础。

Objective To clone the recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit (Ltb) and Actinobacillus actinomycetemcomitans fimbria associative protein (Fap).Methods Two couples of primers were designed for PCR according to the known sequence of ltb and fap. The ltb and fap gene were obtained by amplification PCR technique from plasmid EWD299 of Escherichia coli and Actinobacillus actinomycetemcomitans 310 DNA respectively, and fused them by PCR. The fusion gene ltb-fap were cloning into plasmid pET28a(+). The recombined plasmid pET28a ltb-fap was transformed into Escherichia coli DH5α. The recombinant was screened and identified by restriction enzyme and PCR. The cloned gene was sequenced.Results The ltb-fap about 531bp in size was obtained successfully , and identified by PCR, restrictive enzyme and sequence analysis.Conclusion The vector of pET28a ltb-fap was obstained.