Military Medical Sciences 2014;(3):212-215
doi:10.7644/j.issn.1674-9960.2014.03.012
Construction of expression vector and preparation of antiserum for the undefined gene LOC401296
Liping SHEN ; Hong CHEN ; Xueqing ZHANG ; Jianping ZHOU ; Zhongwu LIN ; Zhidong WANG ; Ying CHEN
Keywords
LOC401296; undefined gene; prokaryotic expression; eukaryotic expression; protein purified; antiserum
Country
China
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 .
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