HBVC区基因克隆和PCR条件优化  

Cloning of HBV Gene of C region and optimization of PCR conditions

作　　者：陈思礼[1] 袁媛[2] 

机构地区：[1]中南民族大学生命科学学院,湖北武汉430074 [2]华中师范大学生命科学学院,湖北武汉430079

出　　处：《中国热带医学》2007年第8期1279-1280,1284,共3页China Tropical Medicine

基　　金：国家民委自然科学基金重点资助项目(MZZ04002);中南民族大学自然科学基金资助项目(YZZ04006)

摘　　要：目的建立PCR优化条件和方法,探讨HBVC区基因在乙型肝炎PCR检测中的应用价值,为荧光定量PCR检测HBV摸索条件。方法将HBV基因组导入DH5α大肠杆菌,SDS碱裂解法小量提取大肠杆菌阳性构建质粒DNA,设计合适引物对HBVC区基因进行PCR扩增,对PCR条件中的退火温度、Mg2+浓度进行了优化,并比较三种不同Taq DNA聚合酶的灵敏度。结果转化的大肠杆菌质粒DNA最佳退火温度为58℃,最佳Mg2+浓度为1.5mM,同时确定天源公司的Biostar Taq DNA聚合酶灵敏度最高,扩增到10-6。结论确立了HBVC区PCR的优化条件,将为后期进行荧光定量PCR检测HBV感染奠定基础以及为探索更有效的检测方法提供实验依据。Objective To establish optimized conditions and methods of PCR and explore the applied value of dectection of HBV C - region gene for diagnosis of hepatitis B by PCR and offereing evidence in dectection d HBV by using fuoreseent quatitative PCR, Methods Genome of HBV was transformed into Escherichia coli DHSaby utilizing CaCl2. SDS alkali cleavage method was used for extracting plasmid DNA in a small amount. HBV gene of C region was amplified by using selfdesigned primer. The anealing temperature and Mg^2＋ concentration were optimized. The sensitivities of three Taq DNA polymerases was compared. Results 58℃ was the best anneaing temperature of the plasmid DNA of Escherichiacoli, the best Mg^2＋ concentration was 1.5mmol/L, and the highest sensitivity of Taq DNA polymerase was as high as 10^-6. Conclusion The optimized conditions has been established that has laid foundation for quafitiative detection of HBV by PCR and helpful for easrly clinical diagnosis of hepatitis B.

关 键 词：乙型肝炎 HBV C区基因 克隆 PCR条件优化 

分 类 号：R512.62[医药卫生—内科学]