Research On The Activity Of ScFv Against HER2-positive Breast Cancer Enhanced By LP-scFv And Rapid Detection Of SARS-CoV-2

This thesis includes two parts.Part 1.Research on the activity of single chain antibody(scFv)against HER2-positive breast cancer enhanced by leader peptides.Part 2.Rapid detection of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2).Part I,LP-scFv enhances anti-HER2 positive breast cancer activity.Breast cancer is the most common cancer in women and is divided into different subtypes,of which 20-30% overexpress the HER2 receptor,known as HER2-positive breast cancer.HER2-positive breast cancer patients tend to present with a poor prognosis and a high risk of relapse.Antibody-targeted therapy significantly improves the prognosis of HER2-positive breast cancer.However,nearly one-third of patients with HER2-positive breast cancer develop resistance to these antibody-based drugs.There are multiple mechanisms leading to drug resistance,the most important of which is the insufficient internalization capacity of antibodies,which limits their therapeutic effect on solid tumors.The promotion of antibody internalization and tissue penetration has become an important challenge to enhance the efficacy of antibody drugs.In this study,LP-scFv drug model was established by coupling anti-HER2 scFv with LP,which had the ability to penetrate the membrane and transport proteins,to improve the effective internalization of scFv and then enhance the anti-tumor activity of scFv.This work would provide drugs and small molecule drug carriers for the anti-activity of HER2-positive breast cancer,provide a reference for the design of novel antibody drugs,and provide a strategy for improving drug resistance.The main contents are: 1)LP-scFv was constructed by conjugating the signal transduction peptide LP with a single chain antibody against HER2,to promote the internalization of scFv;2)Anti-tumor activity of LP-scFv was studied in vitro and in vivo.3)Molecular dynamics analysis of the binding of scFv and LP-scFv with HER2 and the interaction with DPPC phospholipid bilayer,and then dynamic analysis of the molecular mechanism of LP promoting the internalization of scFv.The major conclusions are as follows:1.LP-scfv conjugate was successfully expressed in the E.coli system LP-scFv significantly increased the internalization efficiency of scFv(about 2 times),and maintained scFv cell specificity,HER2 antigen affinity and anticancer activity.By increasing the intracellular dose effect of scFv,LP-scFv enhanced the inhibition of phosphorylation of MAPK-ERK1/2 and PI3K/AKT signaling pathways,showing higher inhibition of cell proliferation and promotion of cell apoptosis in vitro.2.The xenotransplantation model experiments in nude mice in vivo verified that LP1 coupling significantly improved the antitumor activity of scFv,the antitumor effect of LP1-scFv was equivalent to that of Trastuzumab.H&E staining and serum biochemical analysis showed that scFv and LP-scFv had low toxicity,little side effects and good tolerability.3.Molecular dynamics analysis showed that conjugation of LP reduced the binding free energy of scFv with HER2-DPCC and DPPC,which was conducive to promoting the internalization of scFv,providing a theoretical basis for understanding the mechanism of LP promoting the internalization of scFv.4.LP-scFv with simple process has great application potential as a specific drug and small molecule antibody drug carrier model against HER2 positive tumorPart Ⅱ,Rapid detection of SARS-CoV-2.COVID-19 caused by SARS-CoV-2 has become a persistent global pandemic since its outbreak in 2019 and has brought multiple challenges to the global society,economy and health care system.Early diagnosis of viral infection undoubtedly allows rapid intervention,disease management,and substantial control of the rapid spread of the disease.RT-PCR is considered to be the most commonly molecular diagnostic method for diagnosing COVID-19,it is still subject to many limitations,such as a long time(1.5-2 h),the need for specialized training and equipment and so on.It is necessary to develop a reliable,safe,fast diagnostic method for COVID-19.RT-LAMP has the advantages of high sensitivity and strong specificity,short time consumption(within 1 h),visualization,no complex thermal circulatotoy(60-65 °C constant temperature)and no professional operators.This part develops a rapid diagnostic COVID-19 RT-LAMP kit by targeting the S gene or N gene of the SARS-CoV-2 virus,which is helpful for rapid intervention,disease management,and control of the spread of COVID-19.In addition,the extraction of viral RNA becomes the rate-limiting step of RT-LAMP test,and the process of RNA extraction may bring harm to operators,and may also cause secondary contamination,resulting in missed or false detection.Therefore,this study established a closed,visual One-Pot-RT-LAMP kit without the extraction of viral nucleic acid,providing a key technical solution for COVID-19 diagnosis,which is of great significance for epidemic prevention and control.The main contents are: 1)Development of a visual,accurate,reliable,and rapid diagnostic kit for COVID-19 RT-LAMP targeting S gene of SARS-CoV-2 virus;2)A visualization and rapid detection of N gene of SARS-CoV-2 virus by RT-LAMP method;3)Development of a closed,visualized,and rapid one-pot-Rt-LAMP kit for COVID-19 without the extraction of viral nucleic acid based on the RT-LAMP method targeting N gene of SARS-CoV-2 virus.The main conclusions are as follows:1.The RT-LAMP kit for rapid detection of SARS-CoV-2 by targeting the S gene of SARSCoV-2 virus in 35 minutes was designed and developed.The kit had a specificity of 99.00% for clinical sample evaluation.2.The RT-LAMP method for rapid detection of SARS-CoV-2 in 35 minutes was designed and developed by targeting the N gene of SARS-CoV-2 virus.3.Based on the RT-LAMP method for targeting the N gene of SARS-COV-2 virus,The OnePot-RT-LAMP kit was developed to detect SARS-CoV-2 directly from "virus to result" without extracting viral nucleic acid by by adding formamide,achieving the direct and accurate diagnosis of COVID-19 in 45 minutes.The One-Pot-RT-LAMP method had high specificity,and the validation results of 45 clinical samples were completely consistent with the RT-q PCR detection results.4.The developed kits had obtained European CE certification with the certification numbers of DE-CA76-IVD0015-003 and NL-CA002-2020-54368.