| Bovine surfactant protein A(SP-A)is an important lectin protein,which plays an important role in the defense of the lung against the invasion of pathogens.Pasteurella multocida type A is one of the important pathogenic bacteria causing respiratory diseases in cattle,causing huge economic losses to the global cattle industry.Domestic scholars reported that natural BSP-A protein has an inhibitory effect on pathogenic E.coli and other pathogenic bacteria.To investigate whether recombinant BSP-A protein has an inhibitory effect on Pm,In this paper,according to the amino acid sequence of BSP-A protein in Gen Bank,the basic physical and chemical properties of BSP-A protein were analyzed,the prokaryotic expression plasmid was constructed and the recombinant BSP-A protein was purified,and the recombinant BSP-A protein was verified to have agglutination activity and certain antibacterial activity against Pm,and A small amount of natural BSP-A protein was obtained.1.Analysis of physicochemical properties of bovine surfactant protein A and exploration of recombinant protein prokaryotic expression conditions.According to the entry number of bovine surfactant protein A(XP＿00590926.1),the BSP-A is A secreted protein with strong hydrophilicity,isoelectric point of 4.88 and size of 25.54 k Da.The encoding gene of BSP-A protein was synthesized into the plasmid p ET28 a by optimizing the codon of E.coli,but the vector couldn’t express the target protein.p Cold-BSP-A-FSH,p Cold-GST-BSP-A-FSH,p Cold-SUMO-BSP-A-FSH and p Cold-TF-BSP-A-FSH expression plasmids were constructed,After exploring the expression conditions,it was found that only p Cold-TF-BSP-A-FSH expression plasmid could achieve the massive expression of TF-BSP-A-FSH recombinant protein in supernatant under the IPTG condition of 0.8 m M at 16℃.2.Purification and activity study of recombinant bovine surfactant protein A.A large number of TF-BSP-A-FSH recombinant proteins were expressed,and 74 k Da TF-BSP-A-FSH recombinant proteins were purified using Ni-NTA resin.Thrombin cleavage of recombinant proteins was found to be more efficient at 28 °C,9 hours of digestion,and lower non-specific cleavage of proteins of interest.The 26 k Da BSP-A-FSH recombinant protein was purified by Strep-Taction resin.The Tiger Red Plate Agglutination Method detected the agglutination activity of BSP-A-FSH recombinant protein and TF-BSP-A-FSH recombinant protein.In vitro bacteriostatic tests showed that the recombinant TF-BSP-A-FSH protein had a significant inhibitory effect on bovine Pm,while the recombinant BSP-A-FSH protein had a significant inhibitory effect on Pm.The endotoxin-removing recombinant BSP-A protein enhances the phagocytic capacity of bovine macrophages to Pm,indicating that the purified recombinant bovine SP-A protein has bacteriostatic activity against Pm.3.Acquisition of natural SP-A protein in bovineAfter the purified recombinant TF-BSP-A-FSH protein and the purified recombinant BSP-A-FSH protein were emulsified with Freud’s adjuvant,the immune serum was obtained after immunization with Kunming mice,and the immune serum was found to have good repertoire TF-BSP-A-FSH protein and BSP-A-FSH recombinant protein with good reactivity,indicating the successful preparation of BSP-A polyclonal antibodies.The natural SP-A protein of bovine was crudely extracted from the bovine alveolar lavage solution using maltose-Sepharose(MS)gel,and the size of the natural SP-A protein was detected by immune serum as 30 k Da,laying the foundation for the next step of the functional study of BSP-A protein.In this study,the recombinant BSP-A protein was successfully expressed and purified by prokaryotic expression system,and the bacteriostatic activity of the protein on Pm was found,and the BSP-A clone antibody was prepared,which laid a foundation for further studying the function of BSP-A protein. |
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