Construction and
Expression of hTNFα-hTGF3 Fused Gene
CHEN Jian-Jun, SUN Miao, LU Fang, CHEN Chang-Qing and WANG
De-Bao1
( Shanghai Research Center of Biotechnology, the Chinese Academy of
Sciences, Shanghai 200233, China;1Shanghai
Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai
200031, China )
Abstract Human tumor necrosis factor
was fused with hTGF3, the third loop region of hTGFα specifically binding to
EGF receptor, through a flexible linker by recombinant DNA techniques. The
constructed plasmid containing hTNFα-hTGF3 fused gene, pSB92-TLT, was
expressed under the control of PL promoter and after the
induction at 42 ℃ it gave a high level of expression of the fused gene, ranging
in 50%―60% of total bacterial protein. 95% of the expressed product was
inclusion body, and only 5% was soluble. When the fused gene was constructed
into the constitutive plasmid pLC, a new plasmid controlled by PL
promoter but without cIts857, and expressed at 30 ℃, the expression
yield reached 50% and a high proportion of soluble protein, 35% of total
bacterial protein, was obtained. Western blot analysis indicated that the
fusion protein could specifically bind to anti-hTNFα antibody. Comparing to
original hTNFα, the fused hTNFα derivative showed about equal cytotoxicity to
L929 cell line and higher cytotoxicity to some human tumor cell lines. This
shows that the fusion protein TLT may be promising in application.
Key Words hTNFα; TGFα；fusion protein; soluble
overexpression; anti-tumor activity
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