| Backround and Objectives:Epidemiologic studies indicate that there are 18 million sepsis patients every year around the world with a 30%-50% high mortality rate,which can reach 80% if complicated by infectious shock。Thus,to study the pathogenesis of sepsis and explore new medicine and treatment about sepsis is significantly meaningful to sepsis patients.Sepsis can be caused by bacteria, viruses, fungi or parasites, among which gram-negative bacteria is the most common pathogen. Ggram-negative bacteria can multiply rapidly and break down after entering the blood, resulting in release of lipopolysaccharide(LPS), which transmit signals from extracellular to intercellular through TLR4 after a series of signal pathways, followed by the releasement of inflammatory cytokines. TLR4 plays an important role in sepsis as the hub in the process of LPS-induced sepsis. It also becomes an important target in the research and development of therapeutic drugs for sepsis. The present studies on antibodies of TLR4 indicate that mortality was reduced by inhibiting the release of inflammatory factors after suppressing TLR4 in animal experiment, but traditional monoclonal antibodies have weak penetration and high prices which limits its clinical application and exploration to some extent. The nanobodies research based on the discovery of camel heavy chain antibodies provides new approaches for antibody drug exploration in disease diagnosis and treatment.Different from traditional four chain antibodies(two heavy chains and two light chains), camel heavy chain antibodies exist without light chain and show satisfactory antibody activity with VHH single structure with an oval shape, 2.5nm diameter, 4nm height which can also bind with antigens. As its small size, heavy chain antibody VHH is also called nanobody. Because of its small molecule, stable structure, strong tissue penetration, high solubility, easy preparation and low production cost,nanobody has shown good application prospect.Therefore, considering the important role of TLR4 in mediating signaltransmission of sepsis-LPS and combining with the advantages of nanobody in application, our study was designed to build anti-TLR4 nanobodies with phage antibody library technology after building TLR4 extracellular region antigens first,then discuss the protective effect of anti-TLR4 nanobodies to sepsis rats by vitro and vivo experiments on LPS and bacterial sepsis.Methods:1. By analyzing the structure of TLR4, we took the extracellular domain of TLR4 as target gene. Gene amplification and vector construction methods were used to clone the extracellular domain of TLR4 to p ET28 a, a prokaryotic expression vector. After induced by IPTG and purified by Ni ion affinity packing, we finally obtained high purity TLR4 extracellular domain, ex-TLR4.2. Purified ex-TLR4 was chosen as immunogen and camels were taken as the experiment animal. Each camel was immuned once every two weeks by 500 μg ex-TLR4. 4 days after the 6th immune, the antiserum was obtained and detected with ELISA. Camel blood with high titer was collected and PBMC was obtained by density gradient centrifugation. VHH gene was amplified from peripheral blood lymphocytes and then constructed to p CANTAB-5E, a special vector for phage-display-system. The recombinant plasmids were transformed into E.coli TG1 and the volume of the phage library was measured.3. After four rounds of panning by ex-TLR4, 60 single phage plaques were randomly picked and phage ELISA analysis was performed to screen positive clones. Pre-screened positive clones were further screened by TI and TC proteins.Final positive clones were transformed to E.coli HB2151 for the preparation of soluble protein induced by α-lactose. Target proteins(the obtained Nanobody)were purified by Ni2+ metal chelate affinity chromatography and named as TI-Nbs and TC-Nbs.4. In vitro, RAW264.7 cells were pre-stimulated by LPS and TLR4 nano antibody was added to the medium. We took ELISA method to study the effect of nano antibody on the inflammatory factors in RAW264.7 induced by LPS.5. In vivo study, LPS-induced model and Gram negative bacteria induced rat inflammation model were used to study survival rate affected by anti-TLR4nano-antibody.Results:1. TLR441-625 was chosen as the target sequence. We successfully constructed the expression vector and obtained the target protein with superior purity.2. Camels were immunized with purified ex-TLR4. After six immunization, the antibody titer of blood serum reached above 1:128,000. VHH gene was cloned from peripheral blood lymphocytes and successfully constructed to p CANTAB-5E for establishing antibody library. The volume of the phage library was 1.0×109 per ml.3. ex-TLR4 as target protein, the first screen of phage library established above was carried out in four rounds. The enrichment factor of the third round screening reached to 147,the fourth round screening was 3.1, which indicated that phage library for TLR4 has been effectively enriched. After the first screen by ex-TLR4,we got 16 positive clones, and the number of positive clones reduced to 5 for TI and 7 for TC after the second screen. The soluble proteins were prepared fromα-lactose-induced positive E.coli HB2151 clones and the purity of purified target proteins reached above 90%.4. By using LPS-induced cell model of inflammatory, we obtained 3 TI-positive clones and 2 TC-positive clones, which showed the inhibition effect on the releasement of cellular inflammatory factors, and TI nano antibody had a better effect compared to that of TC nano antibody. The synergistic reaction between the TI and TC nano antibody could further reduced the LPS-induced inflammatory response.5. Antibody with the highest neutralization activity determined by in vitro experiment was used to the research on animal test. The results showed that multiple nano antibody administration(TI+TC) had a better effect on animal models of acute inflammation induced by LPS, compared to TI or TC single administration. The level of inflammatory cytokines, such as TNF-α and IL-6,was reduced by the administration of multiple nano antibody administration (TI+TC) on G- septic model rats.Conclusion:This study obtained TLR4 extracellular region antigens and nanobodies against c-terminal and Intermediate domain of TLR4 by protein expression and purification.Vitro and vivo experiment results showed that anti-TLR4 nanobodies could improve animals’ survival rates and inhibite the release of inflammatory factors. Meanwhile,antagonizing both c-terminal and Intermediate domain of TLR4 showed a remarkable effect, which would provide new methods and strategies for clinical treatment of sepsis. |
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