Construction Of Human Naive Fab Library And Characterization Of Anti-Influenza Virus Fab Fragment Generated From The Library

Influenza,as infectious diseases,has a serious impact on people’s health.In addition to vaccine-prevention and some non-specific treatment, specific treatment drug,which can cure virus infectious diseases,has not yet appeared so far.It is an effective way to obtain a large number of antibodies by artificial preparation.In recent years,with phage display technology developing,the whole process of B cells producing antibodies in vivo can be simulated in vitro using phage display technology.It has become a powerful tool for preparation of human antibodies.It is easy to manipulate and have the advantage of high selection capacity.It is also a good way to obtain rare antibodies and to obtain completely humanized McAbs directly.In this project,by using the burgeoning biotechnology of phage display antibody library,we attempt to develop a specific anti-influenza surface antigen monoclonal antibody.This study could be divided into two parts.One is construction and identification of non-immuniezed human antibody libraries.The other one is screening and characterization of anti-influenza virus antibody.（1）:construction and identification of non-immuniezed human antibody librariesLymphocytes were separated from healthy human peripheral blood. The total RNA was extracted and human immunoglobulin genes were amplified by RT-PCR.by this way,we obtained heavy chain genes（Fd fragment） and light chain genes（λ,κ） successfully.The mixed light chain genes and heavy chain genes were cloned into plasmid pComb3XSS,and then E.coli cells XL1-Blue was transformed with pComb3XSS.Rescued by helper phage M13KO7,human Fab combinatorial library with a capacity of about 2.38×10⁷cfu/ml was constructed.This Fab library was identified by clone-PCR and enzyme-digestion,and experimental results are in line with expected results.（2）:screening and characterization of anti-influenza virus antibodyPurified inactivated influenza virus vaccine as the antigen,using solid-phase screening,phage antibody libraries have been enriched after 4 rounds of "adsorption-elution-amplification".Phage antibody harvest rate of the fourth round has been increased about 65 times as against the first round from 1.8×10^-3 to 2.8×10^-5.Bacteria were infected with phage antibody libraries enriched in the final round,and then were spreaded on the plate.One hundred clones were randomly picked,periplasmic proteins were extracted after IPTG-induced expression.Influenza virus as antigen,bacterial culture supernatants and bacterial periplasmic protein were screened using ELISA method.43 positive clones were obtained,10 clones（5、7、16、27、33、44、53、54、72、96）with higher OD value were selected among them.Plasmids were extracted and were digested by double enzyme（SacI and Spel）.We can get the fragment size of 1500bp and 3500bp from three clones（5,53 and 96） out of ten.Bioinformatics analysis was manipulated by biology software,three positive clones had a high degree of sequence homology with human antibody genes.The result of protein sequence alignment showed that three positive clones of the heavy chain（Fd fragment） are IGHV6 subgroup,and the light chain （L chain） separately belongs to IGKV3 subgroup,IGKV1 subgroup and IGKV5 subgroup.The plasmids of three positive clones were extracted and were transformed into non-suppressor E.coli Top10.Transformants were induced by IPTG.We got around 25kD and 50kD bands under non-reducing conditions by SDS-PAGE and Western-blotting analysis, however,we could only get a band of about 25kD under reducing conditions.This result is consistent with literature reports.By ELISA and hemagglutination inhibition test（HI）,soluble Fab antibodies from three positive clones showed binding activity against the surface antigens of influenza virus. The completion of this subject can provide technology platform and ideas for preparation and screening of a variety of antibody in the future. In the meanwhile,for clinical testing of antibodies,especially for prevention and emergency treatment of large-scale influenza,it also lays a solid foundation.