Expression Of Chicken Surfactant Protein A Gene In Escherichia Coli And Its Part Of Antibacterial Activity

Surfactant protein SP-A is a hydrophilic glycolprotein, which is synthesized and secreted by alveolar and bronchiolar epithelial cells, and believed to play an important role in innate immunity. Compared with the study on SP-A of mammals, the study of avian source has not yet been clarified. Since the precursor mRNA of the chicken SP-A (cSP-A) gene sequences was registered in the Genbank by Vitved L, the distribution of cSP-A in tissue cells and its function were not clear. The aim was to get the cSP-A protein by the method of genetic engineering and to study its immune mechanism for the further application in clinical medicine. It mainly includes the following several aspects.Firstly, in accordance with the sequence of the precursor mRNA published in the Genbank, a pair of oligonucleotide primers were designed for amplification. The RT-PCR products were purified, cloned into the pEASY-Blunt vector and sequenced. Through amino acid sequence assembly with bioinformatics software, cSP-A was found belongs to the family of C-type lectins. It is characterized by having a collagen-like domain and a carbohydrate recognition domain (CRD), which has a specific Ca2+-dependent specificity for saccharides and thus the ability tobind complex glycoconjugates on micro-organisms.Secondly, to express the CRD proteins of cSP-A in Escherichia coli (E. coli), and prepare mouse polyclonal antibodies against cSP-A. The cSP-A-CRD gene sequence was synthesized, subcloned into pGEX-6P-1 vector, and expressed in E.Coli BL21(DE3). After the expression products were identified by SDS-PAGE, the gel containing the target protein was cut and further purified. Mice were innoculated with the purified recombinant protein to prepare polyclonal antibodies against cSP-A.The titer and specificity of the polyclonal antibodies were analyzed with indirect ELISA, Western blotting and immunohistochemical fluorescence staining, respectively. The result of indirect ELISA showed that the titer of anti-serum against cSP-A was 105, western blot results demonstrated that the anti-serum from mice specifically reacted with the recombinant cSP-A-CRD, immunohistochemical fluorescence staining and ELISA assy indicated that the polyclonal antibody had the ability to specifically detect cSP-A protein expressed on healthy chicken lung epithelial cells and from healthy chicken bronchoalveolar lavage fluid.Finally, the gel containing the cSP-A protein was cut and further recovered using protein recovery kit. The recovered cSP-A protein were tested by plate agglutination test and agar hole diffusion inhibition test, respectively. The result showed that cSP-A-CRD protein could aggregate poultry Salmonella polyval ent antigen with the presence of calcium ions and exhibit antimicrobial activity against Staphylococcus aureus (ATCC25923),B paratyphoid (CMCC50094), Escherichia coli (CMCC44102) and Pasteurella and other bacteria.These results are helpful for the elucidations of the correlation of structure and functions of cSP-A molecule and may be of potential therapeutic benefit to the poultry with infections.