Screening For The Ligand Proteins Of The Outer Membrane Protein P6 Of Nontypeable Haemophilus Influenzae By Yeast Two-hybrid

Haemophilus influenzae（Hi）is a common gram-negative bacterium,which is usually colonized in human nasopharynx.It can cause a variety of diseases,such as upper respiratory tract infection,meningitis in children,pneumonia,otitis media and acute exacerbation of chronic obstructive pulmonary disease in adults.Nontypeable Haemophilus influenzae（NTHi）and Haemophilus influenzae type b（Hib）are the main subtypes isolated from clinical Haemophilus influenzae infections.With the wide application of Hib vaccine,the incidence rate of Hib has decreased significantly,while the incidence rate of NTHi has increased.In recent years,the resistance of NTHi to a variety of antibiotics is gradually increasing,which brings new challenges to clinical diagnosis and treatment,but so far we still have no effective means to prevent NTHi infections.Therefore,by exploring the pathogenic mechanism of NTHi,it is very important to find a more effective method to prevent and treat NTHi disease.It is found that NTHi outer membrane protein P6 is highly conserved among Hi strains and plays an important role in immune protective response,which has become one of the research focuses of scholars in the domestic and overseas.However,there are few studies on the interaction between protein P6 and host as well as its biological function,and its role in the pathogenesis of NTHi is unclear.In this study,the yeast GAL4 AD fusion c DNA library of human nasopharyngeal epithelial cell NP69 was constructed,and the yeast two-hybrid system was used to find the ligand protein interacting with outer membrane protein P6 in the host,so as to explore the characteristics and biological functions of protein P6,and provide a theoretical basis for in-depth study of the role of protein P6 in NTHi infected host,and interpret the pathogenic mechanism of NTHi from a new perspective.It also provides a new idea for the prevention and treatment of NTHi.First,total RNA was extracted from human nasopharyngeal epithelial cell NP69 with Trizol reagent.The first strand of c DNA was synthesized by Switching Mechanism At 5’end of the RNA Transcript（SMART）technology,and then the high-quality full-length ds c DNA was synthesized by LD-PCR.The fragments which below 200bp in the ds c DNA were removed by CHROMA SPIN-400 purification column.The purified ds c DNA,p GADT7-Rec and denatured Yeastmarker ^TMCarrier DNA were co-transformed into yeast Y187 competent cells to construct NP69 cell yeast GAL4 AD fusion c DNA library with p GADT7-Rec as carrier.The titer of the library was 1.67×10¹⁰CFU/ml,identification of c DNA library polymorphism by PCR,1%agarose gel electrophoresis showed that the size of PCR products was between 0.3～2kb,indicating that the library had good polymorphism.Then,the specific gene P6 fragment was amplified from the genome of NTHi ATCC 49247 by PCR,and the PCR products were digested by Eco R I and Bam H I,and inserted into the bait vector p GBKT7.After colony PCR and double digestion identification,the inserted fragment was sequenced by T7 universal primer on p GBKT7 vector,and the sequencing result was consistent with the nucleotide sequence of P6.The recombinant bait plasmids p GBKT7-P6 was transformed into yeast AH109 and Y187competent cells respectively.It was detected that the bait plasmid neither had self-activation effect nor had toxic effect on yeast cells.The yeast two-hybrid system was carried out with NP69 cell yeast GAL 4 AD fusion c DNA library and p GBKT7-P6.After multiple screening of defective media,β-galactosidase membrane method and PCR to eliminate false positive results,and verified again by backcross experiment.Finally,the positive plasmid was extracted and its base sequence was determined.The proteins encoded by homologous genes were detected in the Gen Bank by BLAST to determine the target genes.The results showed that the protein P6 interacted with multiple gene coding products in the NP69 cell c DNA library,including metallothionein 2A（MT2A）,sorting connexin（SNXs）,proteasome activator PA28 subunitα（PA28α）,interferonγ.In this study,the candidate ligand proteins interacting with protein P6 were successfully screened by yeast two-hybrid system,which laid a foundation for further exploring biological function and molecular behavior of protein P6.