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[Site-specific mutagenesis of murine anti-human TNF-alpha Fab gene and its expression in E.coli]
1Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850,China.
Aim:
To make the gene encoding murine anti-human TNF-alpha (hTNF-alpha) Fab fragment humanized construction and get its soluble expression products.
Methods:
One-step opposite direction PCR method was applied to make the genes of heavy and light chain's variable region of murine antibody site-specific mutagenesis respectively. The mutant genes were cloned into vector pComb3H and induced to express through IPTG.
Results:
It was succeeded that three point mutations were introduced into murine anti-hTNF-alpha monoclonal antibody, involving the replacement of Ser88 with Ala in heavy chain variable region(V (H)) and Glu17 and Lys18 with Asp and Arg, respectively in light chain variable region(V(L)), and that humanized Fab expression vector was constructed. It was confirmed that Fab fragments expressed in supernatant by Western-blot.
Conclusion:
We get soluble expression products of humanized Fab, which provides a sound basis for future research on its activity and purification.
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