Construction of expression vector and preparation of antiserum for the undefined gene LOC401296
10.7644/j.issn.1674-9960.2014.03.012
- VernacularTitle:功能未知基因LOC410296的表达载体构建及小鼠抗血清制备
- Author:
Liping SHEN
;
Hong CHEN
;
Xueqing ZHANG
;
Jianping ZHOU
;
Zhongwu LIN
;
Zhidong WANG
;
Ying CHEN
- Publication Type:Journal Article
- Keywords:
LOC401296;
undefined gene;
prokaryotic expression;
eukaryotic expression;
protein purified;
antiserum
- From:
Military Medical Sciences
2014;(3):212-215
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone the full-length CDS sequence of LOC 401296 , an undefined gene that we found recently, and to obtain both its expression in eukaryotic cells or E.coli and antiserum to LOC401296 protein.Methods The full-length CDS sequence of LOC401296 was amplified by polymerase chain reaction (PCR).Then we established the expression vectors pET28B-LOC401296 and pCMV-Myc-LOC401296 by cloning the full-length CDS sequence into vector PET28B and vector pCMV-Myc respectively.Isopropyl Thiogalactoside (IPTG) was used to induce LOC401296 expression in E.coli.Furthermore,the protein purified and refolded was used to immunize BALB /c mice.The titer of the antiserum collected from immunized mice was identified by ELISA assay and Western blot .Results We cloned the full-length CDS sequence of LOC401296 was successfully .Protein LOC401296 was expressed as was expected and the mouse anti-human LOC401296 antiserum was obtained .The antiserum titer reaching 1∶64 000 was identified by ELISA .Besides, Western blot analysis showed that the antiserum could be used to detect protein LOC 401296 .Conclusion The full-length sequence of LOC401296 is obtained and the mouse anti-human LOC401296 antiserum becomes available .This study can contribut to further research on the undefined gene LOC 401296 .
