1.Structural Characterization Of Group B Streptococcus Virulence Factors 2.Identification Of CDR Loop Sequence Of Monoclonal Antibody

1. Structural Characterization of Group B Streptococcus Virulence FactorsGroup B Streptococcus (GBS) is the leading cause of perinatal and neonatal infection, exits in about 25% of healthy women in the reproductive tract. GBS infection in the uterus increases the risk of pneumonia, sepsis, and meningitis in newborns. At present, antibiotics are used in GBS prevention and treatment. But the emergence of GBS tolerance to antibiotics caused attention for GBS therapy. There is no effective treatment for preventing GBS ascending infection. It is thus urgent to develop new vaccines and antimicrobial agents. The design of inhibitory drugs against GBS virulence protein or its synthetic pathway is a more effective and feasible solution.The genomic sequence of GBS is available. After literature review, we selected ten of GBS virulence factors for structural study including clyA, cylB, cylE in the cyl operator and some surface proteins associated with GBS toxicity. The cyl operator is responsible for GBS haemolysis. The surface protein is indispensable for the growth of GBS and toxicity. We aim to use methods of structural biology to explore the structural basis of these proteins, to find potential druggable sites and to provide theoretical basis for the development of new antimicrobial agents.In summary, we cloned, expressed and purified all of the ten proteins. We further set up crystallization screening for these proteins. Finally, we were able to grow crystals and collect x-ray data and solve initial structures for two of them.2. Identification of CDR Loop Sequence of Monoclonal Antibody against Immune Checkpoint TIGITThe blockade of immune checkpoints by antibodies against co-inhibitory receptors on immune cells has achieved great success in cancer immunotherapy in clinics. The immune checkpoints therapy is different from traditional treatment. It does not target at tumor cells but immune cell inhibitory receptors on the surface of immune cells to activate host antitumor activity. The most well studied checkpoints are the CTLA-4 and PD1, and the antibodies against them have made preliminary success in clinical trials of many cancers. But these antibodies also have limitations and side effects, so it is a globally competitive hot spot to find and validate new inhibitory receptors.TIGIT is a recently discovered inhibitory receptor expressed on NK cells and T cells. Our previous study has showed that TIGIT monoclonal antibody has very good anti-cancer effect in animal models, so TIGIT is a very promising new target for immunotherapy. Anti-mTIGIT mAb is unavailable in the market, so it is urgent to develop efficient and specific anti-mTIGIT mAb.For this research project, we aim to solve the complex structure of TIGIT monoclonal antibody and TIGIT. We aim to identify mTIGIT epitopes and their interaction mode with the antibody CDR loops. We plan to further design mutations of the CDR loop region to optimize the affinity and anti-tumor efficacy of the antibody. In this study, we constructed m(h)TIGIT prokaryotic expression system and purified recombinant TIGIT proteins. In addition, we purified mAbs from the nude mouse ascites and constructed secreted prokaryotic expression system of mAb-Fab region.