幽门螺杆菌cag7-n基因克隆表达及其重组蛋白对BGC-823细胞生物学的影响

目的研究克隆幽门螺杆菌（Helicobacter pylori）cag7-n基因对BGC-823细胞增殖和IL-8分泌的影响.方法采用PCR技术从H.pylori基因组DNA中扩增cag7-n基因片段,构建重组质粒pET-28a-cag7-n,转化大肠杆菌BL21,经Western blot鉴定,纯化的蛋白作用于BGC-823细胞,用MTT法检测蛋白对细胞增殖的影响.结果成功克隆cag7-n基因,经镍柱纯化后可获得纯度为98%的重组蛋白.纯化蛋白浓度在100,150,200μg/mL时,培养时间延长,细胞的增殖受到抑制;在相同的培养时间,细胞的增殖程度随蛋白浓度的增加而降低.结论成功克隆了cag7-n基因,并在大肠杆菌BL21中表达,经过镍柱纯化后得到纯度较高的蛋白,纯化透析后的蛋白与BGC-823细胞共培养,可诱导细胞因子IL-8在mRNA水平上的表达及其对BGC-823细胞增殖的影响.

Cloning Expression of Helicobacter pylori cag7-n and the Influence of Its Recombinant Protein on BGC-823 Cytobiology

Objective To study the influence of Helicobacter pylori, cag7-n genes cloning on BGC-823 cell proliferation and IL-8 secretion. Method PCR technology is used to amplify cag7-n gene segment from DNA of Hpylori genome, to construct recombinant plasmid pET-28a-cag7-n and to transform Escherichia coli BL21. By means of Western blot,the purified protein acts on BGC-823 cell. Then MTT method is used to detect the effects of the protein on the cell proliferation. Result The cag7-n is cloned successfully so that the recombinant protein with 98% purity after purification by Ni column. When the purified protein concentration is 100,150,200μg/mL,the culture time is prolonged and the cell proliferation is inhibited. With the same culture time,the cell proliferation degree reduces as the protein concentration increases. Conclusion The cag7-n gene is successfully cloned and expressed in Escherichia coli BL21. Protein with high purity is obtained by means of nickle colnum purification. The protein after purification dialysis is cultured with BGC-823 cell concurrently,which can induce the expression of cytokines IL-8 on mRNA level. Its effects on BGC-823 cell multiplication is studied initially as well.