改良菌落PCR法筛选、鉴定DHBV部分核衣壳蛋白基因的重组克隆  

Improved colony PCR to screen for and identify recombinant clones carrying partial segments coding for core proteins of the duck hepatitis B virus

作　　者：王玉[1] 于爱莲[1] 姜世金[2] 施鲁笛[1] 陈国敏[1,3] 高鹏[1] 

机构地区：[1]泰山医学院基础医学院,山东泰安271016 [2]山东农业大学动物科技学院,山东泰安271018 [3]泰安八十八医院

出　　处：《中国病原生物学杂志》2012年第4期264-268,共5页Journal of Pathogen Biology

基　　金：山东省优秀中青年科学家科研奖励基金(博士基金)项目(No.2007BS02004);山东省科技发展计划项目(No.2010GNC10914);山东省高等学校科技计划项目(No.J08LF07)

摘　　要：目的优化并验证改良菌落PCR程序的有效性。方法经常规PCR扩增DHBV编码核衣壳蛋白与DNA多聚酶重叠区的部分核苷酸片段(DHBc-dp),纯化特异性扩增产物并连入pMD 18-T载体,转化DH5α感受态细胞后涂布于Amp加倍的LB平板,记为亲代样品(PS),37℃培养12h,室温放置4h～8h。无菌操作挑取细菌转入优化的PCR反应液扩增、鉴定;余菌体室温放置完成二次生长。PS剩余PCR鉴定产物继代转化并涂布LB平板,记为继1代样品(F1S),同样进行菌落PCR鉴定。选取PS和F1S中PCR强阳性样品进行扩增及菌液PCR、质粒PCR、质粒酶切和测序鉴定。结果挑菌后的菌落二次生长成面积扩大的菌苔;优化菌落PCR反应体系保持、甚或提高了原有方法的高度特应性和稳定性;携外源目的片段的重组质粒在菌落PCR反应过程中能保持结构完整,并能在继代转化宿主细胞中忠实复制与扩增。结论改进的菌落PCR程序,继承了既有菌落PCR程序鉴定重组克隆的特异性和高效性,并能有效预防阳性转化子的丢失;同时,在增加优势抗性菌落生长量的前提下有效地抑制了卫星菌落的生长。Objective To verify the efficiency of improved colony PCR.Methods Partial overlapping fragments of core proteins of duck hepatitis B virus（DHBV） and segments coding for DNA polymerase（DHBc-dp） were amplified by polymerase chain reaction（PCR） and the products were purified and cloned into the T vector pMD 18-T.DH5α component cells transformed with a ligation solution were plated onto LB plates with twice as much Amp.Plates marked as the parent sample（PS） were incubated at 37 ℃ for less than 12 h and then left at room temperature for no more than 8h.Sterilized tips were used to ascetically collect predominant resistant colonies.Bacteria were then amplified with PCR solutions and identified while remaining bacteria were left at room temperature to undergo secondary growth.Remaining PS PCR products were transformed into DH5α component cells that were plated on LB plates;these cells were designated filial generation 1（F1S）,and colony PCR was performed.Samples from the PS and F1S that were highly positive according to PCR were amplified and PCR of the bacterial solution and plasmids was performed.Restriction enzyme digestion and DNA sequencing were also performed.Results After colony collection,colonies had extensive secondary growth.PCR of predominant colonies provided equivalent results and even provided better reactivity and consistency than conventional PCR.Recombinant plasmids carrying the insert in question were retained the same structure during colony PCR and were faithfully reproduced and amplified in the F1S.Conclusion Improved colony PCR retained the same specificity and efficiency with regard to recombinant clones as colony PCR had while effectively preventing the loss of positive transformants.The improved method can effectively inhibit the growth of satellite colonies despite the greater growth of predominant resistant colonies.

关 键 词：DHBV 核蛋白基因 菌落PCR 鉴定重组质粒 

分 类 号：S858.32[农业科学—临床兽医学]