北华大学学报（自然科学版）
北華大學學報（自然科學版）
북화대학학보（자연과학판）
JOURNAL OF BEIHUA UNIVERSITY(NATURAL SCIENCE)
2014年
4期
463-467
,共5页
幽门螺杆菌%cag7-n基因%克隆%增殖
幽門螺桿菌%cag7-n基因%剋隆%增殖
유문라간균%cag7-n기인%극륭%증식
Helicobacter pylori%cag7-n genes%cloning%proliferation
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.
目的：研究剋隆幽門螺桿菌( Helicobacter pylori ) cag7-n 基因對 BGC-823細胞增殖和 IL-8分泌的影響.方法採用PCR技術從H. pylori基因組DNA中擴增cag7-n基因片段,構建重組質粒pET-28a-cag7-n,轉化大腸桿菌BL21,經Western blot鑒定,純化的蛋白作用于 BGC-823細胞,用 MTT 法檢測蛋白對細胞增殖的影響.結果成功剋隆cag7-n基因,經鎳柱純化後可穫得純度為98%的重組蛋白.純化蛋白濃度在100,150,200μg/mL時,培養時間延長,細胞的增殖受到抑製；在相同的培養時間,細胞的增殖程度隨蛋白濃度的增加而降低.結論成功剋隆瞭cag7-n基因,併在大腸桿菌BL21中錶達,經過鎳柱純化後得到純度較高的蛋白,純化透析後的蛋白與BGC-823細胞共培養,可誘導細胞因子IL-8在mRNA水平上的錶達及其對BGC-823細胞增殖的影響.
목적：연구극륭유문라간균( Helicobacter pylori ) cag7-n 기인대 BGC-823세포증식화 IL-8분비적영향.방법채용PCR기술종H. pylori기인조DNA중확증cag7-n기인편단,구건중조질립pET-28a-cag7-n,전화대장간균BL21,경Western blot감정,순화적단백작용우 BGC-823세포,용 MTT 법검측단백대세포증식적영향.결과성공극륭cag7-n기인,경얼주순화후가획득순도위98%적중조단백.순화단백농도재100,150,200μg/mL시,배양시간연장,세포적증식수도억제；재상동적배양시간,세포적증식정도수단백농도적증가이강저.결론성공극륭료cag7-n기인,병재대장간균BL21중표체,경과얼주순화후득도순도교고적단백,순화투석후적단백여BGC-823세포공배양,가유도세포인자IL-8재mRNA수평상적표체급기대BGC-823세포증식적영향.
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.
