[1]王丽)△,刘金波),段春燕),等.乙型肝炎核心抗原基因的合成及其原核表达载体的构建[J].郑州大学学报(医学版),2009,(06):1242-1244.
 WANG Li),LIU Jinbo),DUAN Chunyan),et al.Gene synthesis and construction of prokaryotic expression vector of hepatitis B core antigen[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2009,(06):1242-1244.
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乙型肝炎核心抗原基因的合成及其原核表达载体的构建 ()
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2009年06期
页码:
1242-1244
栏目:
论著
出版日期:
2009-11-01

文章信息/Info

Title:
Gene synthesis and construction of prokaryotic expression vector of hepatitis B core antigen
作者:
王丽1)刘金波2)段春燕1)龚舒1)何涛1)
1)泸州医学院基础医学院医学分子生物学实验室 泸州 6460002)郑州大学第一附属医院肛肠外科 郑州 450052
Author(s):
WANG Li1) LIU Jinbo2) DUAN Chunyan1)GONG Shu1) HE Tao1)
1)Laboratory of Medical Molecular Biology, Luzhou Medical College,Luzhou 6460002)Department of Coloanorectal Surgery, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052
关键词:
乙型肝炎病毒核心抗原基因合成定点突变原核表达
Keywords:
hepatitis B core antigengene synthesissitedirected mutationprokaryotic expression
分类号:
R373.2
摘要:
合成乙型肝炎病毒核心抗原(HBcAg)基因并构建原核表达载体。方法:根据大肠杆菌偏嗜性密码子,利用Synthetic Gene Designer软件对HBcAg基因进行密码子优化,分成24条相互重叠的寡核苷酸片段,采用PCR法扩增获得完整的HBcAg基因,经BamHⅠ和XhoⅠ双酶切后定向克隆到pET30a(+),得pET30a(+)HBcAg,经PCR扩增、酶切及测序鉴定后,定点突变法改正错误位点。之后将重组子转入BL21(DE3),IPTG诱导表达,SDSPAGE电泳分析表达情况, Western Blot检测蛋白抗原性。结果:成功构建了重组质粒pET30a(+)HBcAg,SDSPAGE分析显示目的蛋白主要以包涵体的形式在大肠杆菌表达,表达效率64%左右;Western Blot显示表达产物具有较好的抗原性。结论:成功合成HBcAg基因并构建了原核表达载体。
Abstract:
To synthesize gene and construct prokaryotic expression vector of hepatitis B core antigen (HBcAg).Methods:By selecting the biased codon in E. coli, the HBcAg gene was optimized by software of Synthetic Gene Designer and was divided into 24 oligonucleotide fragments. The complete target fragment was synthesized using assembly PCR and cloned into pET30a(+) vector at NdeⅠ/XhoⅠ site. After it was analyzed with restriction endonuclease digestion and DNA sequencing analysis, the gene which contained error was corrected by sitedirected mutagenesis. Then pET30a(+)HBcAg was induced with IPTG. Finally, the expression product was identified with SDSPAGE and Western Blot.Results:The recombinant plasmid of pET30a(+)HBcAg were generated.The target protein existed in form of inclusion body was expressed highly in E. coli,and the antigenicity of the protein was specific.Conclusion: The synthesis of HBcAg gene and construction of prokaryotic expression vector were successful.

参考文献/References:

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备注/Memo

备注/Memo:
△女,1977年生,硕士,讲师,研究方向:感染性疾病的致病机制与防治,Email:1999wangli@163.com
更新日期/Last Update: 2010-05-14