Construction And Screening Of VHH Antibody To Bovine M.tuberculosis

Bovine tuberculosis is a chronic zoonotic infectious diseases of high risk which is a great threat to the public.In 1882,Robert Koch found that the incidence of tuberculosis was mainly due to infection with Mycobacterium tuberculosis,which is a certain proportion caused by M.tuberculosis.Bovine ycobacterium tuberculosis anti-drought and low temperature,with strong resistance,can survive in harsh environments,is make difficulties of the prevention and treatment of tuberculosis.In 1993,the Belgian scientist Hamers found that the camel has a antibody lack of light chain,only by its variable region of the antibody called single domain antibodies,which also known as nanobodies.Nanobodies have the advantages of high affinity,high stability,strong tissue penetration,and high expression.Construction of anti-Mycobacterium tuberculosis VHH antibody library and use phage display technology to obtain high expression,specific strong nanobody,to lay the foundation for the prevention and treatment of tuberculosis.The main contents of this paper are as follows:1.Expression and purification of antigen Ag85B.The recombinant strain of BL21(DE3)(pET28a-ag85B)was used to express and purify Ag85B protein for subsequent experiments.2.Construction of anti-bovine Mycobacterium tuberculosis nanobody library.In this study,BCG immunized camel was used to extract total RNA from camel blood as a template for reverse transcription of cDNA,and nested PCR was used to amplify VHH antibody gene fragment.The target gene fragment and pCANTAB5e vector were digested with NotI and SfiI,and then ligated with T4 ligase.They were transformed into TG1 Escherichia coli to construct the VHH antibody primary library.The VHH antibody library was identified and the library capacity was 7.35 × 106.The results showed that the positive rate of the library was 91.7%.The amino acid sequence was analyzed by DNAMAN,and the amino acid sequence homology was 66.48%.Indicating that the construction of VHH antibody library capacity and diversity can meet the follow-up experiments.3.Screening from anti-bovine Mycobacterium tuberculosis VHH antibody library.Using Ag85B as antigen,using the principle of phage surface display technology,using M13K07 helper phage for"adsorption-elution-amplification" panning.After three rounds of panning the enrichment is 102,indicating that after panning have a great enrichment of specific antibody.A large number of monoclones were randomly selected by protein chip interaction,and positive clones were screened and the affinity of VHH antibody was confirmed.In this study,VHH antibody library was constructed by immunizied camel,and the antibody library was used to screen the VHH antibody.The anti-Ag85B VHH antibody with high affinity was screened by protein chip interaction.To explore the VHH antibody in the diagnosis and treatment of tuberculosis in the course of research laid the foundation.