Preparation Of A Single Chain Antibody Against The Protein P6 Of Nontypeable Haemophilus Influenzae

Nontypeable Haemophilus influenzae(NTHi)belongs to the uncapsulated haemophilus influenzae.And it is a common opportunistic pathogen in clinical infections.It often causes otitis media,sinusitis in infants,acute aggravation of chronic obstructive pulmonary disease in adults,bacterial pneumonia in the elderly and other serious infections,and the incidence of NTHI-related diseases is gradually increasing.At present,the common NTHi detection methods have certain limitations,and it is difficult to meet the requirements of rapid,simple and accurate clinical detection.For example,PCR can only identify whether it is Haemophilus influenzae,but cannot identify the type;Further serological identification is required after isolation and culture of Haemophilus influenzae.The serum used is for capsular type and can only be determined after complete exclusion of(a～f)capsular type.Therefore,an easy,rapid and accurate clinical detection method is urgently needed for direct detection of NTHi.NTHi outer membrane protein P6 is highly conserved,specific and homologous.P6 protein is selected as a specific immunogen and P6 single chain antibody is prepared by phage display technology.Firstly,the genetically engineered bacterium BL21(p GEX-6p-2-P6)was recovered and amplified to induce the expression of GST-P6 fusion protein according to the expression conditions determined in the laboratory.The preliminary results of sodium dodecy sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)were consistent with the expected results.Spectrophotometer-determined absorbance was obtained by adsorbing glutathione microbeads on the fusion label GST,which purified the recombinant protein.Then its absorbance is measured with a spectrophotometer,and the concentration of GST-P6 is calculated according to the formula.The purified protein P6 was taken as antigen and injected into female2-month-old New Zealand white rabbits subcutaneously at multiple points.Indirect enzyme chain immunosorbent assay was used to detect the titer of serum anti-P6 protein antibody.After the titer was qualified(titer no less than 1:10~5),It can be used to build the library later.Total RNA of spleen was extracted and c DNA was synthesized by reverse transcription.Then,using c DNA as template,PCR was used to amplify the heavy and light chain variable region sequences V_Hand V_Lof anti-P6 Ig G antibody,and then Linker sequence was connected to assemble the sc Fv sequence.The sc Fv fragment and p MECS plasmid were cut by Not I and Nco I double enzymes.p MECS-sc Fv recombinant plasmid was constructed by T4 DNA ligase and transformed into Escherichia coli TG1 receptor cells,and then assisted phage M13K07 was added to infect the transformed host bacterium TG1,so as to construct the phage library of influenzae outer membrane P6single chain antibody.It is calculated that the storage capacity of this warehouse is about 2.25×10~8.After the successful construction of phage library,the library should be screened.P6 protein was used as the target antigen to elute the unbound free phage,The phage that binds to the P6 protein was then removed with a competing receptor or pickling,and then elute the recombinant phage specifically bound to the target protein antigen P6 protein,and infect the host cell with the elution phage for propagation and amplification.After3～5 rounds of"adsorption-elution-amplification",highly enriched antibody library with a titer of 7.7×10~8pfu/m L was obtained from phages specifically bound to the target antigen P6 protein.Finally,96 monoclonal strains were randomly selected from the enriched screening plate,and after sc Fv antibody expression induced by IPTG,soluble recombinant sc Fv antibody crude extracts were prepared for ELISA detection.After initial recognition of the sc Fv antibody clone and P6 protein binding good,sequencing analysis revealed that a portion of the gene sequences were accurate,thereby enabling the successful construction of a single-chain phage antibody library.In this study,anti-P6 single chain antibody was successfully prepared by phage display technology,which provided a new idea for further development of simple and accurate NTHi clinical detection method and improvement of NTHi laboratory detection.In the later stage,we will further use the prepared antibodies for the identification of clinical specimens to test the sensitivity and specificity of this method.