邓楚钟,潘耀振
DENG Chuzhong,PAN Yaozhen

• 1:贵州医科大学附院肝胆外科

摘要(Abstract)：

目的:对比同源重组与Gibson Assembly两种方法构建腺病毒载体的效率。方法:增殖并提取AD4DNA,分别用同源重组与Gibson Assembly两种方法,将AD4 DNA镶嵌入p BR322质粒,构建出携带有全长AD4DNA的腺病毒载体,并进行PCR和测序鉴定,对比两种方法构建腺病毒载体的筛选效率。结果:两种方法均能构建出正确的腺病毒载体;筛选同源重组法1 000个菌落中,鉴定360个,仅有1个菌落;Gibson Assembly法112个菌落数中,鉴定其中10个,有3个阳性。结论:Gibson Assembly方法比传统同源重组方法在构建腺病毒载体时更有效。
Objective: To compare the efficiency of traditional homologous recombination method with Gibson Assembly method in constructing adenovirus vector. Methods: AD4 DNA was proliferated and extracted,and inlaid AD4 into the p BR322 DNA plasmid and constructed adenovirus vector with total length of the AD4 DNA by homologous recombination method or Gibson Assembly method,which was amplified by PCR and sequenced for identification. Then,the efficiency of two methods was compared. Results: Both methods could construct the adenovirus vector of right sequence. There were over 1 000 colonies in the plate using homologous recombination method,among which 360 colonies were identified and there was only one positive result. Although there were only 112 colonies in the plate using Gibson Assembly,10 colonies among then were identified and there were three positive results. Conclusion: Gibson Assembly method is more efficient than traditional homologous recombination method in constructing adenovirus vector.

关键词(KeyWords)： 腺病毒;疫苗;重组载体;Gibson Assembly
adenovirus;vaccine;recombination vector;Gibson Assembly

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 邓楚钟,潘耀振
DENG Chuzhong,PAN Yaozhen

DOI: 10.19367/j.cnki.1000-2707.2016.04.018

参考文献(References)：

• [1]Smith JG,Wiethoff CM,Stewart PL,et al.Curr Top Microbiol Immunol[J],Adenovirus,2010(16):195-224.
• [2]Wei CJ,Boyington JC,Mc Tamney PM,et al.Induction of broadly neutralizing H1N1 influenza antibodies by vaccination[J].Science,2010(27):1060-1064.
• [3]Hashem AM,Jaentschke B,Gravel C,et al.Subcutaneous immunization with recombinant adenovirus expressing influenza A nucleoprotein protects mice against lethal viral challenge[J].Hum Vac Immunother,2012(4):425-430.
• [4]Song K,Bolton DL,Wei CJ,et al.Genetic immunization in the lung induces potent local and systemic immune responses[J].PNAS,2010(51):22213-22218.
• [5]Roy CJ,Ault A,Sivasubramani SK,et al.Aerosolized adenovirus-vectored vaccine as an alternative vaccine delivery method[J].Resp Res,2011(1):153-159.
• [6]Frenkel VM,Lengagne R,Gaden F,et al.Adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response[J].J Virol,2002(1):127-135.
• [7]Croyle MA,Cheng X,Wilson JM.Development of stable liquid formulations for adenovirus-based vaccines[J].J Pharmacol Sci,2001(10):2458.
• [8]Tang DC,Zhang JF,DVM HT,et al.Adenovirus as a carrier for the development of influenza virus-free avian influenza vaccines[J].Expert Rev Vaccines,2009(4):469-481.
• [9]Kovesdi I,Hedly SJ.Adenoviral Producer Cells[J].Viruses,2010(2):1681-1703.
• [10]Kim SY,Peng ZH,Kaneda F,et al.Current status of gene therapy in Asia[J].Mo Ther,2008(2):237-243.
• [11]胡朝全,孙诚谊,孙连生,等.重组腺病毒载体含人抑瘤素的扩增及纯化[J].贵阳医学院学报,2004(1):14-17
• [12]Robbins PD,Tahara H,Chivizzani SC.Viral vector for gene therapy[J].Trend in Biotechnology,1998(16):35-40.
• [13]Smith KT,Shepherd AJ,Boyed JE,et al.Gene delivery system for use in gene therapy:an overview of quality assurance and safety issues[J].Gene ther,1996(3):190
• [14]王建功,王晓红,杨俊泉,等.重组人p53腺病毒联合放化疗治疗局部晚期非小细胞肺癌[J].贵阳医学院学报,2014(2):225-228.

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