Construction Of Novel High Throughput Yeast Two-hybrid System And Screening Of Alpaca Antibody Interacting With Major Antigens Of Mycobacterium Tuberculosis

In recent years,with the advances of high-throughput experimental technologies and completion of whole genome sequencing in a variety of species,this would act as a miracle to construct high-throughput and complex proteins interaction network.Yeast-two hybrid technology as the main method of protein interaction,after many years of development,develops in the direction of precision,automation,scale,and high throughput.But until now,the yeast two-hybrid technology is not being used in high-throughput screening of interacting protein,but only using to screen a single protein at a time.The system had made it difficult to meet the demands of large-scale protein interaction networkwhich induce hindrance.TB is a serious infectious disease.At present,tuberculosis prevention and control work faces enormous challenges because of lagging of the basic study which is limited by large-scale protein interaction network research.All of these make us difficult to carry out the study:the large-scale protein interaction research between Mycobacterium tuberculosis.Mycobacterium tuberculosis as a intracellular parasitic bacteria,if therapeutic drug,diagnostic antibodies can be into cellular,they will play a vital role in disease controlling.Nanobody itself has many advantages,such as stable expression,small molecular weight,strong permeability and so on.A new high-throughput yeast two hybrid system based on phiC31/att integrase has been built in our study.Based upon above research,Mycobacterium tuberculosis were used to build protein interaction network between tuberculosis proteins.With the construction of a new high-throughput yeast two hybrid system of many proteins of Mycobacterium tuberculosis antigen screening interaction Alpaca antibody,in order for TB diagnosis and treatment.The major results are as follows:1.Construction of a new high throughput yeast two hybrid system’s recombinant vector.Integron integrase phiC31(EN)has the unique function of integration.It can identify the specific nucleotide sequence ATTP and ATTB.Meanwhile,it specifically targeted the specific nucleotide sequences and their adjacent nucleotide sequences with connected them into a new sequence.The integrated enzyme PhiC31 Integrase sequence and its specific identification of the nucleotide sequence ATTP and ATTB were inserted into the vector pGADT7 and pGBKT7(Clontech)of the GAL4 Matchmaker system,respectively,for construction of a new high throughput yeast two hybrid system’s recombinantvector pGADT7-ATTP-phic31(pmGADT7)and pGBKT7-ATTB-phic31(pmGBKT7).2.Verification of a novel high throughput yeast two hybrid system for positive interaction proteinsThe positive control large T SV40 and mouse p53 protein in GAL4 Matchmaker system were cloned into recombinant vector pmGADT7 and pmGBKT7.The results showed that the expression of protein in the positive control was the same as that of pGBKT7-p53 and pGADT7-T.The integration efficiency of PhiC31 was almost 100%.The positive experimental group was the same as the control group.So,the method was proved to be useful for the known positive interaction protein.3.Application of new high throughput yeast two hybrid technique in the construction Mycobacterium tuberculosisproteins interaction networksConstruction of Mycobacterium tuberculosis membrane protein,secreted protein and lipoprotein bait Library and prey Library.Screening the interacting proteins directlybetween bait library and pery library.The interaction network map of Mycobacterium tuberculosis proteins was obtained.The feasibility of new high throughput yeast two hybrid technology can be used in proteomics.4.Construction of a semi-synthetic antibody library of Alpaca prey,Screening of the Alpacaantibody interacting withtuberculosisESAT6、CFP10、Ag85、HSPX are four major proteins of the Mycobacterium tuberculosis antigen.The AAV virus was constructed on the AAV-CMV vector to express the four proteins simultaneously.Animals infected with the virus are used to obtain specific antibodies against these four antigens in order to get specific CDR3 to Mycobacterium tuberculosis.Semi-synthetic Alpaca antibody prey library was constructed by synthetic Alpaca antibody FR1,CDR1,FR2,CDR2,FR3,FR4 and the obtained antibody specificity of the CDR3 region.Screening of the Alpaca antibody interacting with Mycobacterium tuberculosis.