[Expression of recombinant human alanine aminotransferase (ALT) isoenzyme 2 and preparation of anti-ALT2 monoclonal antibody].
OBJECTIVE
To clone and express human alanine aminotransferase 2 (ALT2) in E.coli Rosetta (DE3), and to prepare monoclonal antibodies(mAb) against ALT2 for diagnostic purpose.


METHODS
The gene encoding alanine aminotransferase 2 (ALT2) was cloned from hepatoma carcinoma cell by RT-PCR, and then inserted into pET28a vector. Recombination plasmids (pET28a-ALT2) were transformed into E.coli BL21. Human ALT2 was expressed as His-tagged fusion proteins and purified by immobilized Ni(2+);-affinity chromatography. The purified fusion ALT2 protein was used as an antigen to prepare mAb against it.


RESULTS
The fusion ALT2 protein was expressed in recombinant E.coli Rosetta (DE3). The enzymatic activity of purified His-tag ALT2 is over 10 000 U/L. Mice were immunized with the purified fusion ALT2 protein, and 5 mAbs against ALT2 were generated.


CONCLUSIONS
Two mAbs with high specificity for ALT2 were selected for further quantitative diagnostic reagent development.
Biopharmaceutical Laboratory, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China. gecui2004@163.com
Cui
C
Ge
Yang-Ling
YL
Xian
Hong-Mei
HM
Liao
Yong-Gang
YG
Bao
Xiang-Zhong
XZ
Ye
Yi-Min
YM
Li
Huan-Zhang
HZ
Xia
D000410:Alanine Transaminase / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D011994:Recombinant Proteins; D000410:Alanine Transaminase