摘要
根据从中国家蚕（Ｂｏｍｂｙｘｍｏｒｉ）蛹中提取的抗菌肽ＣＭＩＶ的氨基酸序列，设计兼顾大肠杆菌和昆虫两种不同体系偏爱密码子的ＣＭＩＶ基因序列；用固相合成法合成６段寡聚核苷酸，通过二步电泳法快速纯化之，复性，连接并克隆到ｐＵＣ９中，转化大肠杆菌ＪＭ１０９；在Ｘ－Ｇａｌ、ＩＰＴＧ、ＡｍｐＬＢ平板上挑选白色克隆，用限制性酶谱法筛选含有合适插入片断的质粒ＤＮＡ；然后通过序列分析得到了含有与设计完全一致的ＣＭＩＶ基因序列的阳性克隆。
Natural cecropins presented strong and broad-spectrum antibacterial and antiviral activities. Butthe amount in vivo was only at a low level even after induction. So we focused on the genetic engineering.The genetic design was successful by the aid of computer. Firstly, in the design of the cecropin CMIV gene weconsidered both E. colt's and insects bias codons according to the amino acid sequence of cecropin CMIV fromBombyx mori. Secondly, the restriction sites EcoR 1 and BamH 1 were arranged at each end of CMv gene,which would facilitate the cloning of CMIv gene. Thirdly, under the control of restriction endonucleases Nde 1and Spe 1, the start codon ATG and the stop codon TAG can be left in the gene or cut off as necessary,which made it possible for both solo and fusion expression of CMIV gene. Fourthly, natural cecropins themselves often comprise Met codons inside the genes. Thus the cleavage of fusion protein CNBr should be troublesome even if the gene expression is successful. In addition, an Asn codon (AAC) was located just beforethe stop codon to guarantee the amidated C-terminus of recombinant cecropin expressed in E. coli.Six oligonucleotides synthesized by Solid-phase were isolated and purified using a two-step electrophoresis (a15K polyacrylamide denaturing gel, including 7M urea, followed by an agarose gel) which is easier and fasterthan the methods previously reported. The different oligonucleotides from crude products were isolated to humiliate peraeffects on the sequential ligation. After fast purification with two-step electrophoresis, the products were annealed, ligated and cloned into pUC9, then transformed into E. coli JM109 strain. Taking theadvantage of the α-complimentary effects, we selected twelve white colonies from X-gal-IPTG-Amp-LBplates. A double-digestion restriction map was made subsequently to verify the recons. In order to get furtherproof, we randomly sampled three of nine recons for sequencing. We observed a recon whose inserted fragment was exactly the same with the design.The procedures of separate ligation and cloning, in spite Of their security, were complicated and boring andsometimes may bring about unexpected results caused by too many steps. Moreover the heterogeneity of themolecules and the unavoidable existence of local secondary structure of different fragments would produce adetrimental effect on the proficiency and correctness of annealing and ligation. Our program solved this problem, making equal complimentary fragments and annealing them separately, then mixing them togetherfor ligation.A mere restriction map can be utilized to select the recons, especially for those genes ligated from several fragments. Customary selecting requires radioactive isotopes for labeling probes, and can do nothing with theminute changes (for example, deletion, insertion or inversion sequence of minor bases). Making use of the restriction endonucleases, we can select tens of clones a day directly, easilty and accurately.
出处
《南京大学学报（自然科学版）》
CSCD
1996年第3期474-478,共5页
Journal of Nanjing University（Natural Science）
基金
江苏省应用基础研究基金
