The Selection Of Random Peptides That Bind To Surface Virulence Protein Of Mycobacterium Tuberculosis (H37Rv) By Phage Display Technology

Tuberculosis(TB)is a chronic infectious disease,caused by Mycobacterium tuberculosis(MTB).There are many types of TB,most of them are the pulmonary tuberculosis.About one-third of the world’s population is currently asymptomatically infected with M.tb.In 2015,about 9 million new cases are found and 1.5 million died because of TB according to WHO(World Health Organization)report.There are approximately 4.99 million pulmonary tuberculosis patients in China,and about 1.3 million new cases were reported last year.China is the second burden country of TB in the world,has 14.3%clinical case compared to worldwide.In recent years,because of the abuse of antibiotics,drug resistant strains of Mycobacterium tuberculosis bring out a series of problem.The surface protein of MTB is closely related to the pathogenicity,lack of surface protein may change the virulence of mycobacterium tuberculosis.The Bacille Calmette-Guerin(BCG)vaccine is the only vaccine proved to be effective against tuberculosis and it remains the most commonly used vaccine worldwide.BCG vaccine is the attenuated live vaccine after more than 200 times continuous passage.The difference of surface proteins between BCG and virulent Mycobacterium tuberculosis is very important to disclose the pathogenic machnism of TB.And the surface proteins of MTB also can be used as idea target to decelop new vaccine and diagnosis reagent.The phenotypic methods of smear microscopy,culture remain the ’gold standard’diagnostics for TB.However,these evaluations are time-consuming and a low detection rate.Some diagnostic tests,X-ray or based on tuberculin,have poor specificity.While other methods with specificity and sensitive for the TB,such as PCR assay and IFN-γ release assays,needs expensive reagents and instruments,and maybe lead to some false positive or false negative diagnoses.The current serological methods cannot reliably distinguish between patients with active disease from BCG-vaccinated and latently infected individuals.There are plenty of antibody tests in the market with poor sensitivity and specificity,and WHO has warned their use in the diagnosis of TB.Therefore,a novel specific method to detect TB with rapid,low cost and sensitive is urgently needed.The genome of Mycobacterium tuberculosis is very large,there are many unknown function proteins,and pathogenic proteins are not clear.Phage display technology has been widely used because of its ability to combine the genotype and phenotype effectively.It can be analysied the theoretical foundation of the interaction mechanism between protein molecules(such as antigens and antibodies,receptor and ligand,enzyme and substrate)by using the target receptors to bio-panning the interacting phage peptides and analyzing the sequence and structure peptides.In this study,we used phage display peptide library technology to select special peptides that could bind to the surface protein of MTB,BCG as a negative target.The selected peptides can be used to develop new diagnosis reagent in next study.Firstly,81nt nucleotides were synthesized,include 7 peptides random sequence and 12 peptides random sequence,and amplified single strand necleotide to double strand.Which were digested by double enzyme and linked into phasmid,pCANTAB 5E.Secondly,the reconstructed phasmid,pCANTAB 5E/7 peptide random sequence and pCANTAB 5E/12 peptide random sequence,was transferred into TG1 by electrotransformation,then cultured on plates and collected all bacteria.It demonstrated 4.08×107 of 7 peptide random sequence and 5.33×108 of 12 peptide random sequence are constructed by bacteria counting,respectively.The diversity of two random sequence librarys were verified by sequenced.Finally,membrane surface proteins of BCG and H37Rv were extracted and coated ELISA plate,respectively.M13K07 was added into two librarys to stimulate expression and generated 7 peptides library and 12 peptides library,respectively.The two peptide librarys were used to biopanning,BCG suface protein as negative target and H37Rv as positive target,the specificity of the recombinant phage was enriched after 3 times washing and amplification.The affinity ability of single peptide was detected by ELISA.Our study lay a foundation for develop a new diagnosis reagent of TB.