Amplification And Analysis V_H And V_Lgenes Of Monoclonal Antibody Against Cysticercus Cellulosae

Cysticercosis cellulosae is a kind of zoonosis caused by the larvae of taenia solium.Cysticercosis cellulosae is widely distributed in china and seriously threating people'health.In most years,preventing and curing this disease depends on medicines,but because of drug resistance appears continuously and the severity of drug residue,they are restricted in clinical application.After the 1990s,the rapid development of the molecular biology promoted the development of antibody researches and genetically engineered antibodies had been excogitated.Among them,single chain antibody is one of most widely recombinant.There are lots of reports in mang fields,but in parasite field,especially,in cysticercosis cellulosae field,there are no reports.This research amplified V_H and V_L of monoclonal antibody against cysticercus cellulosae and layed basises for the construction of ScFv against cysticercus cellulosae. vMethods:Firstly,the hybridoma cells of secreting monoclonal antibody against cysticercus cellulosae were recoveried and cultured till the best growth condition.Agar diffusion test determined activity of hybridoma cell secreting monoclonal antibody against cysticercus cellulosae.Total RNA was extracted from hybridoma cell being high activity by one step Trizol.Then,designed two pairs of primers based on related sequence published in genebank.And amplifying V_H and V_L gene of monoclonal antibody by one step RT-PCR from the extracted total RNA.At last,V_H and V_L were linked with pMD18-T vector,and the cloned plasmids were transformed into JM109.After agarose gel electrophoresis test,the positive clones were identified by blue/white blot,then the positive clones were sequenced by biological company.Resulst:After total RNA were extracted,ultraviolet spectrophotometer and agarose gel electrophoresis determined them.The result showed:A_260/280 was 1.96;the strip of 28sRNA and 18sRNA were clear.So,it showed total RNA were extracted successfully.Amplifying V_H and V_L of monoclonal antibody by one step RT-PCR from the extracted total RNA.The length of V_H and V_L Was about 400bp after PCR products were examined by agarose gel electrophoresis,which corresponded with the characterizations of mouse antibody.It showed we amplified V_H and V_L of monoclonal antibody.V_H and V_L were linked with pMD18-T respectively. After they were determined and reclaimed by agarose gel electrophoresis,the cloned plasmids were transformed into JM109,and selected by blue/white blot.The positive recombinants were identified with common primers by colony PCR.The result of sequencing showed:the full length of V_H was 357bp and encoded 119 amino acids;the full length of V_L was 315bp and encoded 105 amino acids.Homology comparison of nucleotide sequences using Blast program showed:the homology of V_H was 94%and the homology of V_H was 96%,andthe gene sequence of V_H and V_L accorded with the V_H and V_L genes characterization of mouse antibody.But we found there were some changes in complementary determining regions:the V_L gene contained a nucleotide deletion in CDR3,and V_H contained a nucleotide deletion in CDR2.