Construction of directional T vector for gene cloning and expression.
- Author:
Xing ZHONG
1
;
Chao ZHAI
;
Liang CHEN
;
Xiaolan YU
;
Sijing JIANG
;
Hong YAN
;
Dengxiang YANG
;
Lixin MA
Author Information
1. Faculty of Life Sciences, Hubei University, Wuhan 430062, Hubei, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
methods;
DNA, Complementary;
biosynthesis;
genetics;
Escherichia coli;
genetics;
metabolism;
Gene Expression;
genetics;
Genetic Vectors;
genetics;
Humans;
Liver;
chemistry;
Polymerase Chain Reaction;
methods;
Recombinant Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2013;29(4):510-519
- CountryChina
- Language:Chinese
-
Abstract:
Traditional T vector cloning method requires onerous procedures for identifying recombinant, and directional cloning was impossible. In order to overcome these problems, we have devised a directional T vector pETG based on pET-23a(+). For gene cloning, 7 bp partial LacO sequence was introduced into DNA fragment to reconstitute a full length LacO with Bfu I digested T vector. After transformation, blue colonies were selected on LB plate supplemented with X-gal. Restriction enzyme digestion and PCR identification showed that all blue colonies contained the directionally inserted recombinants and the recombinant efficiency was nearly 100%. We have successfully cloned 103 genes from human liver cDNA; in the study complicated procedures for screening of recombinant were not required. Eight pETG clones were picked for protein expression, and all the clones successfully produced corresponding proteins. We demonstrated that the directional T vector was successfully constructed, and it was very suitable for gene cloning and expression.
