Changes Of CSP-A In The Early Stage Of Chick Lung Infection With APEC Anti-APEC Activity Of Recombinant CSP-A

Avian pathogenic Escherichia coli（APEC）is one of the most important pathogens that seriously endanger the healthy development of poultry industry.Currently,chicken surfactant protein A（cSP-A）,a class of hydrophilic membrane surface glycoproteins localized in the chicken respiratory system,is known to its’innate immune functions against pathogens,which is the first line defense against invading pathogens.The protein is divided into chicken surfactant protein A1（cSP-A1,also known as chicken lung lectin）and chicken surfactant protein A2（cSP-A2,formerly known as chicken lung surfactant protein A）,and due to the cumbersome and expensive extraction and purification processes of national cSP-A,hinder the research of cSP-A against pathogens.Therefore,it is very essential to prepare recombinant cSP-A1 and cSP-A2 by genetic engineering technology and to carry out its mechanistic research against APEC.In this study,we established an APEC lung infection model of SPF chickens and explored the expression of cSP-A1 and cSP-A2 in the respiratory system at early infection;prepared cSP-A1 and cSP-A2 recombinant proteins by insect baculovirus expression system and mammalian cell expression system respectively;and carried out the research on the activity of cSP-A1 and cSP-A2 recombinant proteins against APEC in vitro.1、The expression changes of cSP-A1 and cSP-A2 in the early stage of APEC infection in SPF chicks5-day-old SPF chickens were injected intratracheally with 1×10⁶CFU/m L APEC（100μL/chicken）,and the control group was injected with the same amount of sterile PBS,followed by clinical observation and gross dissection at 12 hours post-infection（hpi）and 1,2,and 3 days post-infection（dpi）,and tracheal and lung tissues were collected for histopathological observation and tissue load determination,as well as quantification and localization of cSP-A1 and cSP-A2 using real-time fluorescence quantification PCR（q RT-PCR）and multiplex immunohistochemical staining（m IHC）,and lung tissues at 2 dpi were selected for transcriptome RNA sequencing for further validation.The results of gross dissection and histopathological observations indicated that the APEC lung infection model in chicks was established;the results of q RT-PCR and m IHC revealed that the expressions of cSP-A1 and cSP-A2 in the trachea and lung of the 12 hpi-2 dpi infection group were up-regulated,and the q RT-PCR results at 2dpi were consistent with the RNA sequencing results of the lung transcriptome;the results of bacterial load in tissues showed that,the bacterial load in the lung gradually declined from 1 dpi to 3 dpi of infection groups compared with the control groups.These results indicated that the expression of cSP-A1 and cSP-A2 present a co-upregulation pattern at the initial stage of APEC infection in the lungs of chicks.2、Preparation of cSP-A1 and cSP-A2 recombinant proteins by insect baculovirus expression systemThe Gp64 signal peptide sequence was introduced into the N-terminus of the cSP-A1and cSP-A2 genes by the Overlap PCR technology,and then ligated into the p Fastbac1transfer vector respectively,to construct the p Fastbac-cSP-A1 and p Fastbac-cSP-A2transfer vectors,and then transformed into DH10Bac competent cells,followed by blue-white spot selection to extract Bacmid-cSP-A1 and Bacmid-cSP-A2 recombinant baculovirus plasmid,which were then transfected into sf9 cells to obtain recombinant baculoviruses r Bv-cSP-A1 and r Bv-cSP-A2.Indirect immunofluorescence（IFA）and Western blot analysis showed that cSP-A1 and cSP-A2 recombinant proteins could be expressed in sf9 cells and secreted mainly into the supernatant of the medium in the form of soluble proteins.The protein was purified by Ni²⁺column affinity chromatography,and the size of the obtained cSP-A1 recombinant protein was 25 k Da（monomer）with a yield of 11 mg/L;the size of the cSP-A2 recombinant protein was 60 k Da（dimer）with a yield of 7 mg/L.3、Preparation of cSP-A1 and cSP-A2 recombinant proteins by mammalian cell expression systemThe target genes of cSP-A1 and cSP-A2 were amplified by PCR and ligated into the p KMD-CMV eukaryotic expression vector,respectively,to construct the p KMD-cSP-A1and p KMD-cSP-A2 recombinant expression vectors,and the plasmids were extracted and then transfected transiently into 293F cells,respectively.IFA and Western blot analysis showed that cSP-A1 and cSP-A2 recombinant proteins could be expressed in 293F cells and secreted mainly into the culture medium in the form of soluble proteins.The protein was purified by Ni²⁺column affinity chromatography,and the size of the obtained cSP-A1recombinant protein contained 25 k Da（monomer）and 50 k Da（dimer）in a yield of9 mg/L;the size of the cSP-A2 recombinant protein was 30 k Da（monomer）in a yield of6 mg/L.4、Anti-APEC activity of recombinant cSP-A1 and cSP-A2 in vitroThe effects of recombinant cSP-A1 and cSP-A2 on the growth and viability of APEC and other bacteria were studied by means of bacterial agglutination assays,colony-count assays,and determination of bacterial membrane permeability.The results of bacterial agglutination assays and colony-count assays showed that recombinant cSP-A1 and cSP-A2 had agglutination and inhibition effects on APEC（AE17,AE81）,Staphylococcus aureus（ATCC 25923）,Salmonella pullorum（ATCC 10398）),Salmonella enteritidis（ATCC 13076）,Salmonella paratyphi A（ATCC 9150）and Escherichia coli（CMCC 44102）strains.Furthermore,the agglutination activity of 293F-expressed recombinant protein was higher than that of sf9-expressed recombinant protein.By measuring the membrane permeability of APEC virulent strain AE17,we found that the agglutination of recombinant cSP-A1 and cSP-A2 led to the damage of APEC membrane.In summary,this study established the APEC lung infection model and found that cSP-A1 and cSP-A2 co-upregulated at the early stage（12 hpi-2 dpi）of APEC lung infection in chickens;cSP-A1 and cSP-A2 recombinant proteins were expressed by the insect baculovirus expression system and mammalian cell expression system,respectively;recombinant cSP-A1 and cSP-A2 had agglutination and inhibition effects on APEC virulent strain AE17 and other bacteria,moreover impaired the membrane of APEC virulent strain AE17.