Cloning, Expression and
Antibody Production of the Disintegrin Domain of Human Fertilin β
DING Bei-Bei,
SHEN Wei-Ying, JIANG Chu, WANG Jian, GAO Er-Sheng, SHEN Qing-Xiang*
( National Laboratory of Coutraceptives and Device Research, Shanghai
Institute of Planned Parenthood Research, Shanghai 200032, China
)
Abstract A cDNA for
the disintegrin domain (hf279) was isolated by PCR from human testis
cDNAs. DNA sequencing indicated that hf279 cDNA encoded 93 amino acid
residues, and it was identical with the reported sequence of fertilin
β. An expression plasmid, pGEX hf279, was constructed by inserting hf279
cDNA into plasmid pGEX-4T-2 containing gst gene. The expression plasmid
was introduced into E.coli BL21(DE3) cells and a substantial amount of
soluble fused protein GST-HF93 was obtained by the expression strain HF93/BL21
induced with IPTG. SDS-PAGE analysis revealed that the GST-HF93 fusion protein
had an apparent molecular weight of 38 kD and accumulated up to 50% of
bacterial soluble proteins. The fusion protein was purified by glutathione
S-transferase (GST) Sepharose 4B column (purity＞90%) and digested by thrombin
to obtain the purified HF93 peptide (purity＞80%). Polyclonal antibodies
were obtained from the serum of miceimmunized with purified HF93 which was
isolated by GST Sepharose 4B column and SDS-PAGE. ELISA and Western blot
analysis showed its specificity to HF93. Therefore this antibody can be used in
further studies on the function of HF93.
Key words the disintegrin domain of human
fertilin β； gene cloning； gene expression； E.coli； antibody
*Corresponding author： Tel, 86-21-64049215-3407； Fax, 86-21-64046128； e-mail, [email protected]