<i>LbCpf1</i>基因的原核表达、纯化与体外切割检测 <sup>*</sup>

doi:10.13523/j.cb.2003057

中国生物工程杂志

2020, Vol. 40

Issue (8): 41-48 DOI: 10.13523/j.cb.2003057

技术与方法

LbCpf1基因的原核表达、纯化与体外切割检测 ^*

吕一凡,李更东,薛楠,吕国梁,时邵辉,王春生(

)

东北林业大学生命科学学院哈尔滨 150040

Prokaryotic Expression, Purification of LbCpf1 Protein Gene and in Vitro Cleavage Activity Assay

LV Yi-fan,LI Geng-dong,XUE Nan,LV Guo-liang,SHI Shao-hui,WANG Chun-sheng(

)

College of Life Science, Northeast Forestry University, Harbin 150040, China

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摘要：

目的:为获得具有体外切割活性的LbCpf1蛋白。方法: 将毛螺菌科细菌ND2006(Lachnospiraceae bacterium ND2006)的LbCpf1基因编码区连接至pHis*6(IV),构建原核表达质粒CRISPR-LbCpf1-6*His。将该重组质粒转化BL21(DE3)感受态细胞,IPTG诱导目的蛋白表达,经镍柱亲和层析纯化、透析除盐和凝胶电泳检测等步骤获得重组蛋白,进行体外切割试验鉴定重组蛋白切割活性。结果: 双酶切鉴定和测序结果表明成功构建重组质粒CRISPR-LbCpf1-6*His,经转化后获得含有重组质粒CRISPR-LbCpf1-6*His的BL21(DE3)蛋白表达菌株。将菌株接种于37 ℃,160 r/min,IPTG终浓度为0.5 mmol/L的条件下诱导5 h,最终镍柱纯化除盐后的LbCpf1蛋白终浓度可达400 ng/μl,在体外适宜条件下,该重组蛋白可与成熟的crRNA结合切断标靶DNA。结论: 获得的高纯度LbCpf1蛋白具有体外切割活性,可用于后续基因编辑研究。

关键词： LbCpf1蛋白; 原核表达; 镍柱纯化; 体外切割检测

Abstract:

Compared to Cas9, LbCpf1 has higher targeting specificity and other advantages in eukaryotic cells. Therefore, this study aims to obtain the LbCpf1 protein that is cleaved by in vitro activity. To achieve that, pY016 plasmids containing LbCpf1 gene coding region of Lachnospiraceae bacterium ND2006 were double-enzyme digested to obtain the CRISPR-LbCpf1 gene CDS. Next, the prokaryotic expression plasmid CRISPR-LbCpf1-6*His was constructed by ligating the CRISPR-LbCpf1 gene sequence to the prokaryotic expression vector pHis*6(IV) containing the 6*His tag. Afterwards, high yields of recombinant plasmids were obtained from transformed DH5α competent cells. Then the obtained plasmids were identified by double-enzyme digestion and sequencing, the results of which showed the correct recombinant plasmids were constructed successfully. The correct plasmids were subsequently transformed into E. coli BL21 (DE3) competent cells to generate a BL21(DE3) expression strain containing the recombinant plasmid CRISPR-LbCpf1-6*His, which were then inoculated and cultivated at 37°C, on a 160 r/min shaker. The expression of target gene was induced by IPTG (final concentration 0.5 mmol/L) for 5 hours, and the production was purified by Ni column affinity chromatography, dialysis and desalting, SDS-PAGE gel electrophoresis and other steps to obtain the recombinant protein. The final concentration of the protein can reach approximately 400 ng/μl. Finally, via in vitro cleavage assay, it showed the protein was able to process the pre-crRNA in an appropriate environment and bind to the mature CRISPR RNA (crRNA) to cleave the target DNA in vitro, which verified the recombinant protein cleavage activity. In conclusion, this study provides a method to obtain high-purity LbCpf1 protein, supporting the usage of LbCpf1 in further genetic editing research.

Key words: LbCpf1 Prokaryotic expression Nickel column purification In vitro cleavage activity assay

收稿日期: 2020-03-23 出版日期: 2020-09-10

ZTFLH:

Q78

基金资助: * 黑龙江省大学生创新创业训练计划(201910225232);黑龙江省自然科学基金(C2016012)

通讯作者: 王春生 E-mail: wangchunsheng79@nefu.edu.cn

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引用本文:

吕一凡,李更东,薛楠,吕国梁,时邵辉,王春生. LbCpf1基因的原核表达、纯化与体外切割检测 ^*[J]. 中国生物工程杂志, 2020, 40(8): 41-48.

LV Yi-fan,LI Geng-dong,XUE Nan,LV Guo-liang,SHI Shao-hui,WANG Chun-sheng. Prokaryotic Expression, Purification of LbCpf1 Protein Gene and in Vitro Cleavage Activity Assay. China Biotechnology, 2020, 40(8): 41-48.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2003057 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I8/41

图1 质粒pHis*6经Hind Ⅲ和XhoⅠ双酶切电泳图和质粒图

图2 质粒pY016双酶切产物电泳图和质粒图

表1

表2 靶序列PCR引物

表3 体外切割过程中LbCpf1,crRNA和DNA的摩尔比

图3 CRISPR-LbCpf1-6*His双酶切产物电泳图和质粒图

图4 IPTG诱导LbCpf1的SDS-PAGE分析

图5 重组蛋白LbCpf1的表达类型分析

图6 目的重组蛋白LbCpf1的纯化分析

图7 转录模板和crRNA的琼脂糖凝胶电泳分析

图8 靶序列电泳图和crRNA与靶序列结合的示意图

图9 LbCpf1蛋白的体外切割检测

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