小鼠抗狂犬病毒单克隆抗体轻链重链可变区基因的cDNA克隆

CLONING cDNA OF LIGHT-AND HEAVY-CHAIN VARIABLE REGIONS OF MONOCLONAL ANTIBODY AGAINST RABIES VIRUS

In order to construct a human-mouse chimeric gene of anti-rabies virus monoclonal antibody for producing a chimeric antibody for therapy,the hyb-ridoma cells McAb 2F6 that secreted a McAb against rabies virus with high neutralization titer ( IgG2a,K ) was used as starting material. Total RNA was extracted by guanidine thiocyanate method, and the polyA+-RNA was obtained by oligo-d ( T ) affinity chromatography. According to the 5' end sequences of light and heavy chain constant regions,two complementary oligo-nucleotides heavy chain 3'ATAGGTGACC5'; light chain 3'GACGTGGTTG-5' ) were chemically synthesized and used as primers for directly synthesizing cDNA of light-and-heavy chain variable regions. It was proved that the VK-ds-cDNA and Vr-ds-cDNA were synthesized from incorporation of isotope into first- and second-chain and autoradiography after electropho-resis. The length of these cDNAs was about 1 kb. With dC:dG tailing method, cDNAs were inserted into plasmid pUCl9 and transformed into E.coli JM83, and then recombinants were selected by X-gal and ampicillin. Using probes of immunoglobulin light-and heavy-chain variable regions for in situ hybridization, it was proved that the recombinants contain fragments of light- or heavy-chain variable regions. Further identification is being carricd out.