中国医药生物技术
中國醫藥生物技術
중국의약생물기술
CHINESE MEDICINAL BIOTECHNOLOGY
2013年
4期
241-246
,共6页
定向分子进化%In-Fusion技术%同源区段%高通量克隆
定嚮分子進化%In-Fusion技術%同源區段%高通量剋隆
정향분자진화%In-Fusion기술%동원구단%고통량극륭
Directed molecular evolution%In-Fusion technique%Homologous sequence%High-throughput cloning
目的在目的基因原载体（DNA 模板）与克隆载体相同的条件下，探讨了 PCR 扩增片段（含目的基因）的末端与载体间同源区段长度对 In-Fusion 连接效率的影响，并建立以In-Fusion 技术为基础的高通量克隆方法。方法以糖基水解酶 Lxyl-p1-2（2.4 kb）基因突变库的构建为例，设计引物使目的基因的两端分别与线性化载体末端含有15~200 bp 重叠序列，并通过易错 PCR 方法获得含目的基因的 PCR 扩增片段，运用 In-Fusion 技术将 PCR 扩增片段定向克隆至表达载体 pPIC3.5K 中。结果对于长度大约为2.4 kb 的较大片段 DNA 的克隆， PCR 扩增片段的末端与载体间最适同源区段长度约为100 bp，此时连接效率最高，其阳性重组率高达90%左右，是常规酶切连接法的3倍。与后者相比，该技术将克隆周期由3~4 d 缩短为1~2 d。结论优化的同源区段长度可以显著提高 In-Fusion 技术对于2.4 kb 目的基因与载体的连接效率，该方法尤其适用于定向进化过程中基因突变库的构建。
目的在目的基因原載體（DNA 模闆）與剋隆載體相同的條件下，探討瞭 PCR 擴增片段（含目的基因）的末耑與載體間同源區段長度對 In-Fusion 連接效率的影響，併建立以In-Fusion 技術為基礎的高通量剋隆方法。方法以糖基水解酶 Lxyl-p1-2（2.4 kb）基因突變庫的構建為例，設計引物使目的基因的兩耑分彆與線性化載體末耑含有15~200 bp 重疊序列，併通過易錯 PCR 方法穫得含目的基因的 PCR 擴增片段，運用 In-Fusion 技術將 PCR 擴增片段定嚮剋隆至錶達載體 pPIC3.5K 中。結果對于長度大約為2.4 kb 的較大片段 DNA 的剋隆， PCR 擴增片段的末耑與載體間最適同源區段長度約為100 bp，此時連接效率最高，其暘性重組率高達90%左右，是常規酶切連接法的3倍。與後者相比，該技術將剋隆週期由3~4 d 縮短為1~2 d。結論優化的同源區段長度可以顯著提高 In-Fusion 技術對于2.4 kb 目的基因與載體的連接效率，該方法尤其適用于定嚮進化過程中基因突變庫的構建。
목적재목적기인원재체（DNA 모판）여극륭재체상동적조건하，탐토료 PCR 확증편단（함목적기인）적말단여재체간동원구단장도대 In-Fusion 련접효솔적영향，병건립이In-Fusion 기술위기출적고통량극륭방법。방법이당기수해매 Lxyl-p1-2（2.4 kb）기인돌변고적구건위례，설계인물사목적기인적량단분별여선성화재체말단함유15~200 bp 중첩서렬，병통과역착 PCR 방법획득함목적기인적 PCR 확증편단，운용 In-Fusion 기술장 PCR 확증편단정향극륭지표체재체 pPIC3.5K 중。결과대우장도대약위2.4 kb 적교대편단 DNA 적극륭， PCR 확증편단적말단여재체간최괄동원구단장도약위100 bp，차시련접효솔최고，기양성중조솔고체90%좌우，시상규매절련접법적3배。여후자상비，해기술장극륭주기유3~4 d 축단위1~2 d。결론우화적동원구단장도가이현저제고 In-Fusion 기술대우2.4 kb 목적기인여재체적련접효솔，해방법우기괄용우정향진화과정중기인돌변고적구건。
Objective To explore the effect of length of homologous sequence between the ends of PCR amplicon (harboring the targeted gene) and the vector on the In-Fusion recombination efficiency, when the template vector is the same as the cloning vector, and to establish an efficient high-throughput cloning method based on In-Fusion technique. Methods With construction of the mutant library of the glycoside hydrolase Lxyl-p1-2 (2.4 kb) as an example, different primers were designed on the basis of the various positions of the vector. The PCR fragments (each harboring the targeted gene) were obtained by error-prone PCR. Each fragment contained two over-lapping sequences (15 - 200 bp in length) at the two ends with each of the corresponding ends of the linearized vector. These PCR amplicons were directionally cloned into the vector pPIC3.5K by In-Fusion technique. Results For the cloning of the 2.4 kb DNA fragments by the In-Fusion method, the highest DNA ligation efficacy was obtained when the over-lapping sequence between the PCR fragments and the linearized vector at any end was 100 bp in length, with the highest recombination rate of around 90%. The recombination rate was three times higher than that of the conventional restriction enzyme cloning method. Moreover, the cloning period was drastically shortened from 3 - 4 d of the conventional method to 1 - 2 d of the present method. Conclusion The optimized length of homologous sequence obtained in this paper may significantly increase the ligation efficacy between the 2.4 kb targeted gene and the vector when the In-Fusion technique was applied. The method is particularly suitable to construct the mutant library in the study of directed evolution.
