CAJ | 학술논문

建立一种简单的重组质粒构建方法——二步PCR，不需要限制性内切酶对基因和载体进行酶切。第一步常规PCR中，用正向引物（IF）和反向杂合引物（IR）扩增黑曲霉木聚糖酶Xyn基因，IF与基因特异性匹配，IR引物3’端与基因序列匹配、5’端含有19 bp pET20b一端正链序列；第二步反向PCR中，以扩增的Xyn基因作为正向引物，其3’端含有19 bp与环状pET20b模板退火、并在高保真Q5 DNA聚合酶作用下延伸，与模板另一端互补的反向引物VR协同作用，重组质粒通过PCR扩增出来。反向PCR产物经T4 DNA连接酶环化，并经DpnI限制性内切酶消解处理，可以直接转化BL21（DE3）感受态细胞。用这种方法将622、639、1125、1209 bp大小的基因重组pET20b中，显示出有较广泛的普适性。
건립일충간단적중조질립구건방법——이보PCR，불수요한제성내절매대기인화재체진행매절。제일보상규PCR중，용정향인물（IF）화반향잡합인물（IR）확증흑곡매목취당매Xyn기인，IF여기인특이성필배，IR인물3’단여기인서렬필배、5’단함유19 bp pET20b일단정련서렬；제이보반향PCR중，이확증적Xyn기인작위정향인물，기3’단함유19 bp여배상pET20b모판퇴화、병재고보진Q5 DNA취합매작용하연신，여모판령일단호보적반향인물VR협동작용，중조질립통과PCR확증출래。반향PCR산물경T4 DNA련접매배화，병경DpnI한제성내절매소해처리，가이직접전화BL21（DE3）감수태세포。용저충방법장622、639、1125、1209 bp대소적기인중조pET20b중，현시출유교엄범적보괄성。
A simple restriction enzyme-free method,step-reverse PCR,was developed to construct recombinant plasmid in two steps. An Aspergillus niger xylanase gene(Xyn),for example,was amplified in the first step with a forward primer(IF)and a chimeric reverse primer(IR). IR 3’-end was complementary to Xyn gene,and its 5’-end contained a 19 bp fragment of one end of pET20b(+). In the second reverse PCR step,the amplified Xyn 19 bp 3’-end served as the forward primer to anneal to and extend along circular pET20b template,and the recombinant plasmid was amplified by cooperation with an inverse primer(VR)complementary to pET20b at the other end. After treatment with T4 DNA ligase and restriction enzyme DpnI,the PCR product was directly transformed home-made competent BL21(DE3)E. coli cell. Generality was checked by cloning various sizes of genes(622,639,1125,1209 bp)into pET20b.