The Role Of Chicken TGF-?1 In The Secondary Infection Of Escherichia Coli Induced By H9N2 Type Avian Influenza Virus

H9N2 subtype low pathogenic avian influenza virus(LPAIV)is widely distributed all over the world.The secondary simultaneous infection with other bacteria,especially E.coli,has caused great economic losses to poultry industry.Although H9N2 virus infection has been proved to promote bacterial infection,its mechanism is not clear.Through the preliminary study in the laboratory,TGF-?1,integrin,cortical protein,cadherin,adhesion spot protein,fibronectin and other proteins that may be related to bacterial adhesion were preliminarily screened.TGF-?1,as an immunomodulator and proinflammatory factor,can mediate the adhesion of some bacteria.At present,most of the researches on TGF-?1 are focused on human source,but there is no report on the relationship between chicken TGF-?1(The homology of chicken TGF-?1 with human amino acid sequence is only about 60%)and avian pathogenic bacteria infection.These results suggest that TGF-?1 protein may play an important role in the secondary E.coli infection induced by H9N2 virus.Chicken TGF-?1protein may play an important role in the secondary E.coli infection induced by H9N2 virus,but the more detailed mechanism is still unclear.Therefore,this study takes the relationship between virus,host and bacteria as the main line to reveal the host factors involved in the process of secondary bacterial infection induced by H9N2 AIV in chicken,providing a new theoretical basis for explaining the synergistic pathogenesis between influenza virus and bacteria,and providing a molecular target for the research and development of anti-infective drugs.This study is divided into the following three parts:1.Analysis of the ability and characteristics of H9N2 subtype AIV to inducing E.coli to adhere to host cellsChicken embryo fibroblasts(DF-1)were cultured on 6-well cell plates to monolayer cells.H9N2 virus was inoculated into 3 holes random Ly,and the other 3 uninfected holes were used as control.After 24 hours,APEC was inoculated into six holes of the cell plate,incubated at 4? for 1.5 hours,scraped off the cells,and the number of APEC cells was measured by coating method.In vivo,30 2-week-old SPF chickens were fed with aseptic isolators and 15 SPF chickens were inoculated with H9N2 virus via nasal cavity.7 days after inoculation,APEC was inoculated as the experimental group.The other 15 uninfected chickens were inoculated with APEC as the control group.The lung tissues of the control group and the experimental group were fixed overnight in the freshly prepared 4%paraformaldehyde solution,and then embedded in paraffin.The histological changes were observed by hematoxylin and eosin paraffin staining.The infection of H9N2 virus was identified by immunofluorescence staining.Two groups of chicken lung tissue abrasives(200?l)were coated on LB nutrient agar plate,and the total number of colonies was counted.The results of immunofluorescence staining showed that the chicken lung was infected with H9N2 AIV,and the collapse of pulmonary alveoli and the aggregation of inflammatory cells were found on the pathological sections.In vitro and in vivo experiments showed that H9N2 virus was beneficial to the adhesion of APEC.2.The effect of H9N2 virus infection on the expression and activity of TGF-?1 in chickensWestern blot analysis and qRT-PCR showed that the transcription and expression of TGF-?1 protein in DF-1 cells infected with H9N2 virus increased;According to the fact that the proliferation of mink lung epithelial cells can be strongly inhibited by TGF-?1,the activity of TGF-?1 in the supernatant of DF-1 cells infected with H9N2 virus was detected by the proliferation inhibition test of mink lung epithelial cells.The supernatant of DF-1 cells inoculated with H9N2 virus for 12 hours,24 hours,36 hours and 48 hours was dropped into96 well cell culture plate with mink lung epithelial cells,cultured in incubator for24 hours.The growth inhibition rate of mink lung epithelial cells was calculated after fixation,staining,dissolution and OD value determination,so as to reflect the activity change of TGF-?1 in the supernatant of cells infected with H9N2 virus.The results showed that the activity of TGF-?1 in the supernatant of DF-1 cells increased in a time-dependent manner after H9N2 virus infection.At the same time,one week old SPF chickens were inoculated with H9N2 virus in the nasal cavity.The expression of TGF-?1 in lung abrasive fluid of chickens infected with H9N2 virus was detected by ELISA kit and q RT-PCR.3.The effect of chicken TGF-?1 protein on the adhesion of E.coli to host cells.The reverse transcripts of RNA from chicken lung tissue infected with H9N2 virus were used as templates.TGF-?1 was amplified by PCR with TGF-?1 specific primers.The target fragment was connected with p MD18-T vector.The sequenced p MD18-T-TGF-?1 vector was digested by two restriction endonucleases,Xho I and Eco R I.After the gel was recovered,the correct target gene was sequenced and identified by sequencing,and then the pc DNA3.1(+)overexpression vector was used two kinds of restriction endonucleases,Xho I and Eco R I,were digested by two enzymes,and then the products were linked by tool enzyme T4 ligase,and then transformed into E.coli.The successful plasmids were transfected into DF-1 cells by liposomes.The results showed that the adhesion of APEC to cells in the overexpressed cells was significantly higher than that of the control group.At the same time,the target gene and p ET-28a(+)prokaryotic expression vector were connected by the same method,and the constructed recombinant prokaryotic expression plasmid was transformed into the BL21(DE3)competent cell which were inoculated in liquid medium and cultured at 37? for 8 hours with IPTG.Centrifuging what was left at 4?.After 2houes,4hours,6hours and 8 hours of IPTG induction then disrupts the bacteria.It was found that the size of 44.5k Da was the target band in protein gel chromatography,and no such band appeared in blank control pore by SDS-PAGE and Western Blot blot.The results showed that the size of the band in the positive pore was consistent with the target protein.Western blot detected the same size of the strip,which was consistent with that detected in SDS-PAGE.The expressed protein was purified by nickel column,and then detected by SDS-PAGE.At this time,there was only a single band with a size of 44.5 k Da.The target protein was added to DF-1 cells in vitro,and then APEC was inoculated.After incubation,centrifugation and washing,the number of APEC adhering to the cells was detected.The results showed that the number of E.coli adhering to the cells with exogenous TGF-?1 protein was more than the cells without TGF-?1 protein,and the number of bacteria increased with the increase of TGF-?1 protein dose.These results indicate that H9N2 AIV infection is beneficial to the adhesion of APEC to host cells,and TGF-?1 host protein is related to the adhesion of APEC.Next,by adding TGF-?1 receptor inhibitors and si RNA interference,we reverse verified that the decreased expression of TGF-?1 protein weakened the adhesion ability of APEC to cells,which indicated that the role of TGF-?1protein in the adhesion process of E.coli was crucial.