Anti-lung Single-chain Antibody Fusion Construct And Expression Of The Human Thymosin ¦Á1

Thymosin α1, a 28-amino-acid peptide, is widely used in the diagnosis and treatment of hepatitis B, hepatitis C, tumors, immune-deficiency syndrome, et al. Many researches attest to the interest paid to its fusion protein with other active factor, such as GST, thioredoxin and IFN α-2b.LC-1 single-chain Fv (LC-1 ScFv) is a monoclonal antibody obtained from the secretion of LC-1, which was later constructed based on recombinant phage displayed techniques. ScFv is the smallest segment being able to interact with antigen. Because of its small molecular weight and lack of Fc, it has a stronger force of penetration and immunogenecity than conventional antigen. To date, the expression of fusion protein between Tal and ScFv has not been reported. In theory, this fusion protein should has both of the activity of Tal and antibody. The fusion protein can be used for the monitoring and treating of lung cancer, and be apllied to the research of the acceptor of certain cell factor.The total RNAs were isolated from LC-1 monocolony cells, which have a high secretory volume and activity, and transfered into cDNAs later. According to ScFv gene, the variable regions of heavy chain (V_H) and of light chain (V_L) were cloned through PCR, respectively. ScFv gene was formed with the SOE ligation of V_H and modified V_L. The ScFv gene amplified by PCR was cloned into plasmid pET-22b(+) between the sites for Nco I and Sal I .At the same time, the Tal gene was cloned into pUCm-T vector, and subsequent t-ly inserted into the C-terminal of ScFv gene between the sites for Sal I and Hind III, then transformed into E. coli BL21 cell line. It had a perfect expression with a 5h's induction using 0.5mmol/L IPTG at 3h0℃, in which the recombinant protein shared 30% of the total proteins of the induced cells.