Construction, Screening And Identification Of The Fab Phage Antibody Library Against B-lymphoma

AIM:Phage antibody library is the technique to display the antibody genes on phage vector by molecular cloning and obtain the specific antibody genes with antigen affinity selection and phage amplification.By this technique,various monoclonal antibody genes including Fab,ScFv,dsFv and diabody can be produced.This study aims to construct and screen the specific Fab phage antibody library against Raji cell strain in human B-lymphoma.METHODS:In this study,BALB/c mice were immunized with Raji cells and antisera were titrated by ELISA.Following the demonstration of sufficient antibody titer, total RNA was got from splenic lymphocytes.Light chainκgenes and heavy chain Fd fragments were amplified by RT-PCR.After restrictive digestion with SacI/XbaI and XhoI/SpeI,light chainκgenes and heavy chain Fd fragments were successively inserted into the phagemid vector pComb3H-SS and then electroporated into E.coli XL1-Blue.The specific Fab phage antibody library against Raji cell strain in human B-lymphoma was constructed by infection with helper phage VCSM13.Following five rounds of "adsorption-elution-amplification" biopanning with Raji cells coated on six-well plate, random clones from the last round plate were selected to express the soluble antibodies in E.coli XL1-Blue and the binding activities with antigens were identified by ELISA.The positive clones were further sequenced and the specificity of antibodies was identified by Western blot analysis.RESULTS:(1)After 1.5%agarose gel electrophoresis,about 700bp fragements of the IgG Fd and light chainκgenes were amplified as expected.(2)The Fab library was constructed in two steps.In the first step,the amplified products of the light chainκgenes were mixed,digested,purified and conjugated with phagemid vector pComb3H-SS,and then electroporated into E.coli XL1-Blue to construct the light chainκlibrary.The quantity of the transformants was 4.5×10~7 and the recombination rate reached 100%by SacI/XbaI digestion.So the practical size of the light chainκlibrary was approximately 4.5×10~7.In the second step,the light chainκgene library was amplified and the phagemid named pComb3H-S+κwas extracted.Phagemid pComb3H-S+κand heavy chain Fd fragements were both digested by XhoI/SpeI,and then conjugated and electroporated into XL1-Blue to construct Fab gene library.The quantity of the transformants reached 3.5×10~7.The Fd insertion rate was 78%by XhoI/SpeI digestion,and the Fab insertion rate was 86%by SacI/SpeI digestion.So the practical size of Fab library was about 3.5×10~7×78%×86%,i.e. 2.35×10~7.(3)The phage antibody library infected with helper phage VCSM13 was enriched by five rounds of "adsorption-etution-amplification" biopanning with solid phase antigen of Raji cells.The yield of input-output was gradually increased during the screening,which demonstrated that the phage antibodies against membrane antigens on Raji cells were enriched obviously and the final yield of phage antibody was increased 240 times than that of the first round.Sixty-five monoclones were randomly selected to produce the soluble Fab antibodies and eight positive clones which specifically recognized Raji cell strain were isolated.Sequence analysis of two positive clones showed that the variable light domains(V_L)were both 94%homologous in amino acid with the registered mouse Ig kappa variable germline in GenBank.The variable heavy domains(V_H)were 89%and 92%homologous in amino acid with the registered mouse Ig heavy variable chain germline in GenBank.Western blot results indicated that the soluble products of positive clones specifically recognized the membrane antigens on Raji cells.CONCLUSION:The Fab phage antibody library against Raji cell strain of B-lymphoma were successfully constructed by the phage display technique and specific antibodies against membrane antigens on Raji cells were obtained and identified,which will provide an experimental foundation for the further investigation of B-lymphoma immunotherapy.