Construction Of Human Single Chain Antibodies Library Using Ribosome Displayand Screening Of Scfvs Against HCV NS5B Protein

Hepatitis C virus (HCV) is the causal agent of hepatitis C for infectious diseases and is transmitted mainly through blood-brone, chronic infection is the distinct feature. There are 170 to 200 million people suffered from HCV infection. In China, the number of infected people is about 40 million. About 75 to 85% of HCV infection would proceed into with chronic hepatitis that will be further developed to liver cirrhosis and liver cancer. Currently, HCV infection has become a severe global public health problem. So far, the standard treatment for HCV isα-interferon in combination with ribavirin, only approximately 50% of patients could acquire sustained virological response. Furthermore, These drugs are expensive and have significant side effects. So it is urgent to develop more safe and effective antiviral drugs and biological products.The NS5B of HCV, RNA dependent RNA polymerase (RdRp), is responsible for viral RNA synthesis, while there are no counterpart enzymes in cells, NS5B polymerase has been regarded as an ideal target for anti-HCV therapy. In recent years, a variety of nucleoside and non-nucleoside inhibitors against NS5B have been designed. However, clinical trials showed these inhibitors were easy to produce resistant viral mutants, which affected the further application of inhibitors. Due to the requirement of a certain spatial conformation convertion of NS5B polymerase during the replication of HCV genomic RNA, the conformation inhibitors has become an another important aspect against NS5B polymerase activity.Single-chain antibody (ScFv) can express in target cells delivered by vectors and bind specificly with its antigen, which can block the conformational transition of the target molecules into and change its subcellular localization, that result in the key molecule selective "knock out". This study the humanized single-chain antibody of anti-NS5B protein was screened from constructed human single-chain antibody library by ribosome display, which may inhibit the spatial configuration change of NS5B protein and may provide new hope for the further development of anti-HCV biological agents.【Aim】Due to RNA polymerase NS5B playing a critical role in the HCV RNA replication, NS5B protein was regarded as ideal drug target. The total mRNA has been extracted from the PBLs of HCV positive patients to construct scFv gene library. The scFv library was panned against NS5B protein using the ribosome display technology which is completely in vitro screening and has a large library. The primarily biological function of isolated scFv against NS5B was assessed.【Methods】1. Using the primers designed by bioinformatics software, the ns5bΔ21 gene was amplified with BB7 replicon as template. The purified ns5bΔ21 gene was digested by BamHⅠand HindⅢand inserted into pRSETA with T4 ligase. The positive recombinant plasmid named pRSETA-ns5bΔ21 was transformed into E. coli BL21. The NS5B polymerase was expressed in the transformants after inducing by IPTG and soluble analysis. The antigenicity and specificity of NS5B protein was identified by Western-blot analysis. The targeted protein was purified with Ni2+-NTA affinity chromatography column and electroeluting methods. The purified NS5B protein was used to prepare polyclonal antibody from immunized mice.2. Blood samples were drawed from six HCV positive patients. Total mRNA was isolated from peripheral blood lymphocytes (PBLs) using the Trizol Kit. The quality of total mRNA was detected by formaldehyde denaturing gel electrophoresis. A full set of heavy chain variable region (VH) and light chain variable (VL) gene was amplified from the full-length cDNA. The products of VH and VL were used to construct human single chain antibody (ScFv) gene library by ribosome display. The recombinant plasmid ScFv-pMD18T was transformed into E. coli BL21. Total 60 clones were picked randomly from plate. The 31 positive clones, which was confirmed by restrictive enzyme digestion analysis, were sent for sequencing.3. The purified NS5B protein was coupled with magnetic beads as target antigen. The vitro transcription and translation of ScFv DNA library was performed in rabbit reticulocyte lysate (TNT) system to generate scFv-ribosome-mRNA (ARM) complexes. The ARM complexes were screened using NS5B as coupled antigen. Isolated ARM was used to extract RNA using Trizol Kit. Then the recovered mRNA was transcripted and amplified for producing the display template DNA. The PCR products were performed for next round of the cycle. The specific scFv gene of the anti-NS5B was screened from scFv gene liabrary after three cycles and was sequenced.4. The ScFv gene of anti-NS5B was amplified with positive recombinant plasmid Anti-NS5B-ScFv-pMD18T. The purified scFv gene was digested by NdeⅠand BamHⅠand inserted into prokaryotic expression vector pET16b with T4 ligase. The positive recombinant plasmid named Anti-NS5B-ScFv-pET16b was transformed into E. coli BL21. The scFv against NS5B was expressed after inducing by IPTG. The solubility of the scFv was analysed and identified by SDS-PAGE and Western-blot analysis.【Results】1. Cloning,Expression and Purification of the truncated HCV ns5b gene The enzyme restriction analysis and sequencing results showed that the recombinant plasmid pRSETA-ns5bΔ21 was successfully constructed. The new band was observed on SDS-PAGE after the transformants were induced by IPTG. The Western-blot results showed that the expressed NS5B protein has good specificity and antigenity. The truncated NS5B protein was purified through Ni2+-NTA column and electroeluting methods. The titer of anti-NS5B serum prepared from immunized mice was 1:3000.2. Construction of human svFv library by ribosome displayTotal RNA was extracted from PBLs of six HCV-positive patients. The 28s and 18s rRNA bands were observed on formaldehyde denaturing gel electrophoresis, which showed that the quality of the total RNA is good. The VH and VL gene were amplified though overlapping extension. The purified PCR products were assembled though (Gly4Ser)3 linker. The scFv DNA library was successfully constructed. The sequencing analysis results of 31 positive clones indicated that the diversity of the svFv library is good and the library capacity, is about 7.86×1012.3. Screening of the scFv against NS5B proteinThe ScFv DNA library was performed in vitro transcription and translation system. The results of SDS-PAGE demonstrated that the scFv-ribosome-mRNA complexes were effectively formed. The scFv gene of against NS5B was screened from ARM complexes with coupled-antigen and sequencing results are correct.4. Prokaryotic expression and identification of scFv of anti-NS5BThe recombinant plasmid ScFv-pET16b was successfully constructed and transformed into E. coli BL21. The new band was observed in the SDS-PAGE gel whose size is about 27kD after the transformants were induced by IPTG. Western-blot results further showed that the expressed scFv has good binding activity with NS5B protein.【Conclusions】Humanized scFv library was constructed by using ribosome display technology and the scFv against HCV NS5B activity was screened and identified. This study will provide new ideas and lay a foundation for treatment of HCV studies by using human single chain antibody.