Construction And Bioscreening Of A Camelid VHH Nanobody Library Against CTGF,and Efficiency On Lipopolysaccharide(LPS)-induced Acute Lung Injury In Mice

IntroductionThe novel heavy chain antibodies lack of the CHI domain were consisted of only one heavy chain including a variable domain and two constant regions of CH2 and CH3. Compared with VH-VL pairing of conventional antibodies, the variable domain of the H chain of heavy chain antibody (VHH) binds antigen without requiring domain pairing. And the VHH is capable of antigen binding. The discovery that camelids produce functional antibodies devoid of light chains by Hamers-Casterman in 1993 brought a further breakthrough. After that, the bactrian camels, dromedaries, and llamas were also confirmed of the presence of heavy chain antibody.The VHH nanobody with a molecular size of only 15 kDa consists one single domain, and is much smaller in comparison to the single-chain variable fragment fragments (30 kDa), Fab fragments (60 kDa) or the whole antibodies (150 kDa). Nanobody is the smallest antibody with antigen binding efficiency. Nanobody has several advantages for biotechnological applications.Nanobody is easy for heterogeneous expression in eukaryotic or prokaryotic systems and is more soluble. Nanobody is more stable and it could be stored in RT. Also, it was easy for humanized reformation. Therefore, nanobody as a novel genetic engineering antibody is of important significance and has great prospects for application in disease diagnosis and treatment.In our previous study, the humanized single-chain variable fragment (scFv) antibody against the CTGF was obtained from a phage display human antibody library,and it was shown that it may play a potential role in attenuating pulmonary fibrosis in mice. It has been shown that the recombinant anti-CTGF scFv antibody can neutralize the biological activity of CTGF and decrease the differentiation of fibroblast into myofibroblast.However, pulmonary fibrosis is the end-stage of the lung disease caused by multiple biological, physical or chemical factors. And it is generally believed that inflammation or injury occurred in the early stage. Hence, drug intervention at the early stage to prevent or attenuate the inflammation would be promising for fibrosis-related diseases.ObjectivesIn this study, we aimed to construct a camelid VHH antibody library against CTGF-C terminal (CT) protein and obtain the anti-CTGF VHH nanobody by bioscreening. The anti-CTGF VHH nanobody was expressed and purified from E.coli cells. The efficiency of the VHH nanobody were further evaluated in mice model of lipopolysaccharide (LPS)-induced acute lung injury.Methods1. Recombinant CTGF-C terminal (CT) Antigen Preparation. CTGF-C terminal (CT) gene with optimized codons was synthesized to construct the expression vector pET32a- TrxA-His-CTGF-C, which was transformed into E. coli BL21 (DE3)for expression. After the enterokinase cleavage reaction of the fused protein, CTGF-C was purified by Ni-NTA protein purification system. SDS-PAGE, ELISA and Western Blot were applied to evaluate the immunoreactivity of the CTGF-C. Also, the CCK-8 and transwell experiments were performed to observe its activity in cell proliferation and migration. 2. Construction of a camelid VHH antibody library against CTGF.Total RNA previously isolated from the peripheral blood lym-phocyte pools of a bactrian camel immunized with recombinant CTGF-C terminal (CT) was reverse-transcribed into cDNA. The VHH repertoire was obtained by two rounds of nested PCR and then inserted to pHEN2 vector. The constructed recombinant vectors were applied to E. coli cells for transformation with electroporing. Purified phage particles were the CTGF-specific phage library of bactrian camel single domain antibody fragments. 3.Bio-panning of CTGF specific VHH antibody. The constructed antibody library was applied for bio-panning with CTGF-C terminal (CT) coated onto the 96-wells plates.And four rounds of bio-panning were applied with the selection strength increased in each round. Finally, the eluted phage with affinity to CTGF-C terminal (CT) were collected. 4. The expression and purification of the CTGF VHH nanobody and its preliminary activity study. The nanobody fused with a His-tag was cloned into pET plasmid to construct the expression vector. The recombinant was transformed into E.coli BL21 (DE3) and the protein expression were induced by IPTG. Nanobody was purified by high affinity Ni-NTA column. SDS-PAGE and ELISA were applied to evaluate the immunoreactivity of the nanobody. Besides, we tested its activity on theBEAS-2B, MRC-5 and HLEF cells. 5. Efficiency of CTGF VHH nanobody on lipopolysaccharide (LPS)-induced acute lung injury in mice. Mice were injected with lipopolysaccharide via trachea to establish acute lung injury model following by injected with CTGF VHH nanobody via intravenous. Mice were randomly separated into five groups (NS, LPS, DEX, VHH-1 and VHH-2). At 24h and 48h after intratrachealed, the bronchoalveolar lavage fluid (BALF) were collected for cells counting and classification, and pulmonary tissues were sampled for HE stain or Masson stain. Efficiency of nanobody on lipopolysaccharide-induced acute lung inflammation was evaluated.Results1. Recombinant CTGF-C terminal (CT) Antigen Preparation. The soluble expression of the TrxA-His-CTGF-C fusion protein was induced by IPTG in E.coli.After purification by affinity chromatography and cleavage by enterokinase, the purity of the CTGF-C terminal (CT) reached over 95%. And it exhibited an excellent immunoreactivity by ELISA assay. The western blot analysis further proved that the protein was CTGF-C. Also, CTGF-C (>60 ng/ml) increased HUVEC and HK-2 cell proliferation and cell migration. 2. Construction of a camelid VHH antibody library against CTGF. After the final immunization, the total IgG titer of immunized serum reached 1:12,800 indicating the success of raising immunogenic response in the camel.In the second nested PCR,only the VHH gene was retrieved with a size of ?400 bp.VHH DNAs were digested with NcoI and NotI and then inserted into pHEN2. The constructed recombinant vector was applied to E.coli cells for transformation. The positive clones were approximately 1.6×109 and the insertion rate of the VHH gene was 93%.The VHH genes differed from the three CDR regions and the calculated repertoire reached about 1.49×109. The DNA sequencing of twenty random picked clones results were diversed from each other. 3. Bio-panning of CTGF specific VHH antibody. After four rounds of panning against CTGF-C terminal (CT),12 colonies were randomly chosen for sequencing. The sequencing results indicated that nine clones shared a consensus sequence which named Nbl (GenBank Access:NO.KX428017). 4. The expression and purification of the CTGF VHH nanobody and its preliminary activity study. The recombinant nanobody fusion protein was well expressed as a soluble protein after IPTG induction in E.coli. After purification by affinity chromatography, SDS-PAGE analysis showed that more than 95% purity nanobody was obtained. Also, the ELISA results indicated that it exhibited an excellent immunoreactivity to CTGF-C terminal (CT). The cell proliferation induced by CTGF-C terminal (CT) was inhibited when the cells were treated with the VHH nanobody in BEAS-2B,MRC-5 and HLEF cells. 5. Efficiency of CTGF VHH nanobody on lipopolysaccharide(LPS)-induced acute lung injury in mice. In this study, we successfully established the acute lung injury mice model. It showed that the tracheal epithelial cells were irregularly arranged,shedding or atrophied and inflammatory cells were observed in the lung tissue. HE and Masson stain showed that the VHH groups had a lower degree of alveolitis in lung in comparison with LPS group. At 24h and 48h after treatment of nanobody, the total cells and neutrophils in BALF in the VHH-1 group and VHH-2 group were evidently lower than LPS group (P<0.05). Our data showed that the LPS- iduced up-regulation of TNF-? and IL-1? in BALF and serum were also remarkably inhibited by the VHH nanobody. A lower expression of P65 in lung tissues in the VHH-1 group and VHH-2 group was found by western blot. However, the significant change of Pho-P65 level was not observed.ConclusionThe results indicated the successful construction of the CTGF VHH nanobody library with high quality and diversity. Then, a VHH antibody that could specifically recognize CTGF-C terminal (CT) was successfully selected from the camel library. The cell proliferation induced by CTGF-C terminal (CT) was inhibited when the cells were treated with the VHH nanobody in BEAS-2B, MRC-5 and HLEF cells. The severity of lung injury in the LPS-induced model was attenuated by treatment with CTGF VHH antibody. Also, it might result from the reduced production of P65 and down regulation of NF-?B pathway.