[Cloning and sequence analysis of light chain gene of human antibody against HBV pre-S2]

To obtain the gene of light chain of human antibody against HBV pre-S2.

A human antibody against HBV pre-S2 displayed on phage was obtained from the constructed human immunoglobulin phage displaying library through four rounds panning of "adherence-elution-amplification" with Pre-S2 (120aa-145aa) short peptide synthesized and help phage. The positive clone of anti-pre-s2 was characterized by ELISA competitive inhibition assay. The plasmid from the positive clone was amplified by PCR with designed oligonucleotide primers of kaph light chain. The light chain fragment was amplified with PCR and plasmid pUC18 digested by restriction endonuclease SacI & XbaI, respectively, then it was subcloned into the plasmid pUC18 and was transformed into E.coli XL1-blue sensitized by CaCl2. The recombinant plasmid pUC18-kaph was sequenced.

The competitive inhibition rate of the positive clone of anti-pre-S2 was 65% at 1:50 dilution. Sequence was compared with data in gene bank through internet and analyzed with Blast. According to the sequence and ELISA competitive inhibition assay the integral gene of kaph light chain of human antibody against HBV-PreS2 was obtained. Its variable region located on 1-328bp including three hypervariable regions (82-105bp, 145~177bp, 274~309bp) and four frame regions (1-81bp, 106-144bp, 178-273bp, 310-328bp). Its constant region was located on 329-645bp.

The HBV pre-S2 kaph light chain is screened from phage antibody displaying library by antigen-antibody special response.