Expression Of Recombinant CSP-A And Evaluation Of Its Antibacterial Effect On Pathogenic Bacteria

Chicken Surfactant Protein A(cSP-A)is a hydrophilic glycoprotein secreted by chicken lung epithelial cells,which has a non-specific anti-pathogenic effect.Natural cSP-A protein has a good antibacterial effect on a variety of gram-negative bacteria and gram-positive bacteria,but the extraction and preparation process is cumbersome,the yield is low,and the cost is expensive.The ordinary prokaryotic expression and eukaryotic expression of cSP-A with good biological activity are difficult to achieve the expected results.In this paper,conect the isoleucine zipper structure in Escherichia coli,and use the interleukin 2 promoter in 293T cells and the lentiviral system to establelish a tablele cell line to express cSP-A.The obtained recombinant cSP-A protein was evaluated for the antibacterial effect,which laid the foundation for the follow-up study of the antibacterial mechanism and clinical application of cSP-A.1 Use isoleucine zipper structure to express cSP-A in E.coliIn the study,the N-terminus of cSP-A and the synthetic isoleucine zipper structure were connected by two short leucine peptides to construct recombin--ant IZ-Leu2-cSP-A,and then it was inserted it into the p ET-32a vector to construct a recombinant the vector called p ET-32a-IZ-Leu2-cSP-A.Then the plasmid wastransformed into BL21(DE3)competent cells,and different concentrations of isopropylthiogalactoside(IPTG)were added at 16°C,30°C,37°C to induce the expression for 24 h,12 h,and 6 h respectively.The bacterial liquid was lysed by ultrasound,purified by denaturation and renaturation,and tested by SDS-PAGE and Western Blot.The results showed that the optimal conditions for cSP-A protein were induction temperature of 30°C,IPTG concentratio of 0.5 mmol/L,and cSP-A fusion protein size of 44 k Da.Western Blot results showed that the protein could be recognized by cSP-A monoclonal antibody.The antibacterial test showed that,compared with the kanamycin positive control,the recombinant cSP-A expressed in E.coli had no activities to inhibit the tested bacteria.2 Use interleukin-2 promoter in 293T cells to transienly express cSP-AIn this study,the whole cSP-A gene was inserted into p KMD-IL plasmid,which contains the interleukin-2 promoter,and the p KMD-IL-cSP-A recombinant vectorand was constructed.The recombinant plasmid was transformed into DH5?competent cells.The plasmid without endotoxin was extracted and transfected into 293T cells for transient expression and identification,then the expression was amplified in 200 m L culture system and purified by nickel column affinity chromatography.The SDS-PAGE results showed that the size of cSP-A protein was 24 k Da,which was consistent with the expectation.Western Blot results showed that the protein can be recognized by cSP-A monoclonal antibody.The protein concentration of sample value,which was determined by the BCA method was calculated as 0.4 mg/m L,0.35 mg/m L,and 2 mg/m L respectively.The results of antibacterial test showed that cSP-A protein had a certain antibacterial effect on Salmonella enteric subsp ATCC13076 under the action of Ca²⁺(2.5mmol/L)compared with kanamycin positive control,but had no obvious antibacterial effect on other indicator bacteria.The results of the micro-antibacterial test showed that the minimum inhibitory concentration of cSP-A protein against Salmonella enteric subsp ATCC13076 under the action of Ca²⁺(2.5mmol/L)was 100?g/m L.3 Establelishment of cell line stablly expressing cSP-A and preliminary evaluation of pathogenic bacteriaIn this study,the cSP-A fragment was inserted into the lentiviral vector p CDH-S-His which carries the green fluorescent protein(GFP),to construct the p CDH-S-GFP-His-cSP-A transfer vector.The p CDH-S-GFP-His-cSP-A plasmid and the helper plasmids p MD2.G and p SPX2.G were co-transfected into 293T cells in a certain proportion,named p CDH-GFP-cSP-A;An empty vector control group named p CDH-GFP was set up.The packaged lentiviral particles infected with 293T cells for48 h.The expression of GFP in the cells was observed.The positive cells were screened under pressure with 2.5?g/m L puromycin to screen out the293T-GFP-cSP-A cell line and 293T-GFP cell line.The results showed that the fluorescence quantity of both groups could reach about 80%after 48 hours'transfection.The titers of p CDH-GFP-cSP-A group and p CDH-GFP group were 1.43×10⁶ TU/m L and 2.58×10⁷ TU/m L.RT-PCR results showed that the cSP-A m RNA could be expressed in the 293T-GFP-cSP-A cell line,and it could not be expressed in the 293T-GFP cell line.Western blot results showed that the293T-GFP-cSP-A cell line had cSP-A protein expression,while the 293T-GFP cell line didn't.The results of the antibacterial test showed that cSP-A protein had a certain antibacterial effect on Escherichia coli CMCC44102,Salmonella paratyphi A ATCC9150,and Salmonella enteric subsp ATCC13076 under the action of Ca²⁺(2.5mmol/L)vcompared with kanamycin positive control.In this experiment,the recombinant cSP-A protein was expressed in the three expression systems of Escherichia coli,293T cells and lentiviral system.The recombinant cSP-A expressed in Escherichia coli has no antibacterial activity.The transient expression of cSP-A in 293T cells is only effective against enteritis.Salmonella enteric subsp ATCC13076 has a certain antibacterial effect.The recombinant cSP-A protein expressed in the lentiviral expression system has a certain antibacterial effect against Escherichia coli CMCC44102,Salmonella paratyphoid ATCC9150,and Salmonella enteric subsp ATCC13076.The aim of this study is to explore the expression of proteins through different expression systems and select recombinant cSP-A proteins that have inhibitory effects on a variety of pathogenic bacteria.The recombinant cSP-A gene is cloned by PCR,and three expression plasmids,prokaryotic,293T cell and lentivirus expression system are constructed.The recombinant cSP-A is induced from the three expression systems.A series of bacteriostatic tests are conducted to evaluate the bacteriostatic effect of cSP-A protein on pathogenic bacteria,in order to further explore the support for large-scale production of poultry cSP-A protein through genetic engineering technology,and lay a foundation for the research and development of poultry lung surfactant preparations.