Preparation Of OmpA Polyclonal Antibody And Its Application In The Sample Pretreatment Of Molecular Diagnostic Of Escherichia Coli

Molecular diagnostic technology plays a very important role in current clinical microbial detection,which can be achieved by in vitro amplification of target microbial DNA.However,due to the relatively complex matrix in clinical samples and low bacterial abundance,using it directly as a template for molecular diagnosis may lead to test results false positive or false negative.Therefore,we need a sample pretreatment method that integrates separation,purification,and enrichment to process clinical samples,so as to improve the purity and abundance of target microorganisms in the treated samples and facilitate the subsequent molecular diagnostic detection work.Immuno-magnetic separation technology(IMS)is a recently emerging sample preparation method.Based on the principle of immunological separation,the antibody of the target microorganism is characterized on magnetic beads to form Immonumagnetic Beads(IB).Through the magnetic separation can effectively in a complex matrix by antigen-antibody combination mechanism to separation,purification and enrichment of the target microorganisms.Outer membrane protein A(OmpA),as a protein widely present on the surface of Escherichia coli(E.coli),can be modified to magnetic beads to form IB-OmpA,which can theoretically be used to targeted recognition of E.coli.In this study,IMS combined with molecular diagnostic technology was used to the sample pretreatment of molecular diagnostic of E.coli.The main research contents and results of this paper are as follows:Ⅰ.Construction and purification of OmpA prokaryotic expression vector:According to the E.coli OmpA gene sequence found on NCBI,Primer 5 software was used to design nested PCR primers.The OmpA gene was amplified by nested PCR,the expression vector p ET-32a-OmpA was constructed.IPTG was used to induce the expression vector to express OmpA,and SDS-PAGE results showed that the recombinant protein was expressed in the inclusion body.Inclusion body proteins were treated with 8 M urea and purified by affinity chromatography to obtain purified recombinant OmpA.Ⅱ.Preparation of anti-OmpA polyclonal antibody: The purified OmpA recombinant protein was used as the antigen,mixed with Freund’s adjuvant,and then immunized to Balb/c mice.After four immunizations,blood was drawn to separate the serum.The titer tested by ELISA was 256000.E.coli,Klebsiella pneumoniae,Pseudomonas aeruginosa and Acinetobacter baumannii were used as antigens to evaluate whether anti-OmpA antiserum can be used to capture Gram-negative bacteria.The results show that anti-OmpA antiserum can detect all E.coli strains,and show a certain cross-reactivity to other gram-negative bacteria tested,but there are differences in strains.Ⅲ.Evaluation of IB-OmpA-PCR sample pretreatment method: Anti-OmpA polyclonal antibody was conjugated to magnetic beads(IB-OmpA)and put it into the liquid sample containing E.coli Through the ability of antigen-antibody specific binding,IB-OmpA can capture E.coli in the liquid samples.Afterwards,the captured material is concentrated and separated by applying an external magnetic field,and the product is tested by PCR technology.The results show that IB-OmpA-PCR can be used to enrich and detect E.coli,and its detection sensitivity is about 100 times higher than unused processing methods.Further verification showed that the method could enrich E.coli in various complex liquid matrices(blood,urine,juice,etc.).In this study,the recombinant OmpA protein were expressed and purified.Then use it to prepare anti-OmpA polyclonal antibodies and the feasibility of binding to E.coli were evaluated.Finally,the E.coli molecular diagnosis method of IB-OmpA-PCR was established by combining IMS technology and PCR technology.The IB-OmpA-PCR method can quickly and effectively isolate and enrich E.coli from liquid matrix without need traditional culture medium sort microorganisms,which improves the detection sensitivity and reduces the influence of matrix on detection,and provides a new idea for the pretreatment method of E.coli in clinical samples.