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目的 :构建小鼠“REKENcDNA 0 61 0 0 0 6H1 0”基因表达载体pPRO 6AB ,了解此表达载体在大肠杆菌中表达的情况。 方法 :从克隆有“REKENcDNA 0 61 0 0 0 6H1 0”基因cDNA的载体pUCm 6AB中切取长约 1 1kb的“REKENcDNA 0 61 0 0 0 6H1 0”基因cDNA ,将它克隆到原核表达载体pPROTet E的多克隆位点中。以限制性内切酶酶切分析的方法对重组克隆进行初步鉴定 ,以测序法对重组子进行验证。在确证得到了预期的表达载体pPRO 6AB后 ,以氯化钙转染法将表达载体导入大肠杆菌BL2 1PRO中作蛋白表达分析。蛋白分析方法采用SDS变性聚丙烯酰胺凝胶电泳 ,所用胶浓度为 1 0 % ,交联度为 3 3 %。电泳结束后利用考马丝亮蓝染色法进行染色。 结果 :得到了预期的“REKENcDNA 0 61 0 0 0 6H1 0”基因原核表达载体pPRO 6AB ;“REKENcDNA 0 61 0 0 0 6H1 0”基因成功地在大肠杆菌中实现了表达。 结论 :我们成功地构建了小鼠“REKENcDNA 0 61 0 0 0 6H1 0”基因表达载体pPRO 6AB ,在大肠杆菌中成功地进行了“REKENcDNA 0 61 0 0 0 6H1 0”基因的表达 ,从而为进一步研究“REKENcDNA 0 61 0 0 0 6H1 0”基因编码蛋白的结构和功能 ,探讨“REKENcDNA 0 61 0 0 0 6H1 0”基因在糖尿病肾病发生、发展过程中的可能作用创造了条件
OBJECTIVE: To construct a mouse “REKENcDNA 0 61 0 0 0 6H1 0” gene expression vector pPRO 6AB to understand the expression of this vector in E. coli. METHODS: The cDNA of “REKEN cDNA 0 61 0 0 0 0 6H1 0” of about 11 kb in length was cloned from the vector pUCm 6AB with the cDNA of “REKEN cDNA 0 61 0 0 0 6H1 0” cloned into prokaryotic expression vector pPROTet E In multiple cloning sites. The recombinant clones were identified by restriction endonuclease digestion and the recombinant was confirmed by sequencing. After confirmed that the expected expression vector pPRO 6AB was obtained, the expression vector was introduced into E. coli BL21PRO by calcium chloride transfection to make protein expression analysis. Protein analysis method using SDS denaturing polyacrylamide gel electrophoresis, the gel concentration of 10%, the degree of crosslinking of 33%. After electrophoresis, staining was performed using Coomassie brilliant blue staining. RESULTS: The prokaryotic expression vector pPRO 6AB of “REKENcDNA 0 61 0 0 0 6H1 0” gene was obtained. The “REKEN cDNA 0 61 0 0 0 6H1 0” gene was successfully expressed in E. coli. CONCLUSION: We successfully constructed the mouse “REKENcDNA 0 61 0 0 0 6H1 0” gene expression vector pPRO6AB and successfully expressed the “REKENcDNA 0 61 0 0 0 6H1 0” gene in E. coli so as to further To study the structure and function of “REKENcDNA 0 61 0 0 0 6H1 0” gene coding protein and to explore the possible role of “REKENcDNA 0 61 0 0 0 6H1 0” gene in the occurrence and development of diabetic nephropathy