Development Of Monoclonal Antibodies Against Human Neuropilin 1 B1b2 Domain And Construction Of Single Chain Antibody Prokaryotic Expression Bacteria

Neurolpilin, a mμltifunctional receptor non-tyrosine kinase, binds the class 3 semaphorins (SEMA 3) and vascular endothelial growth factor (VEGF). It was first discovered that neurolpilin mediates the development and growth of nervous system, NRP1 and NRP2 play an essential role in axon growth and guidance. More and more evidence shows that these receptors have an important role in tumor progression. The expression of NRP is increased and NRP has a great relationship with tumor progression and prognosis in a variety of tumors. By influence or direct effect on tumor cells on angiogenesis, neuropilin indirectly affect tumor development by angiogenesis or directly having an effect on tumor cells. The significance of NRP for cancer treatment is great. In this study,we cloned and expressed NRP1 blb2 domain (HuNRP1b1b2) and have got monoclonal antibodies (McAbs) against recombinant HuNRP1b1b2 protein. We extracted the antibody’s heavy and light chains from biologically active hybridoma cell lines by genetic engineering techniques. Finally we got single chain antibody prokaryotic expression bacteria.Objective:Construction of anti-HuNRPlblb2 single chain fragment variable phage display library, lay the foundation for further study of the biological activity HuNRP1b1b2.Methods:①Cloned and expressed HuNRP1b1b2②Prepared anti-HuNRP1b1b2monoclonal antibody and analyzed its biological characteristics③The gene of anti-HuNRPlblb2antibody’ Heavy Variable and Light Variable were amplified by RT-PCR from total RNA isolated from hybridoma.④Overlap PCR (overlap-PCR, RT-PCR) method to get the ScFv.⑤Clone the ScFv into the phagemid vector pCANTAB5E by double restriction enzyme digestion.⑥The recombinant plasmid was transformed into E. coli TG1.ResμLts: ① Successfμ Lly obteined the protein of the HuNRP1b1b2 ② Prepared anti-HuNRPlblb2monoclonal antibody and analyzed its biological characteristics ③ SuccessfμLly obtained hybridoma VH, VL gene fragment sequences. ④ Obtained fμLl-length sequence of a single chain antibody ScFv, and the length was 750bp. ⑤ recombinant plasmid pCANTAB5E-ScFV were constructed successfμLly. ⑥ The recombinant plasmid was transformed into E. coli TG1.Conclusion:HuNRP1b1b2-ScFV was successfμLly constructed,lay the basis for further research of the HuNRP1b1b2 biological activity.