Cloning, Expression And Purification Of Recombinant CARDs TX Protein And Polyclonal Antibody Preparation

Objective:As a pathogen of SARS, Mycoplasma pneumoniae not only can cause acute and chronic respiratory infections also can cause, including the skin, joints, central nervous system and a series of extrapulmonary disease. However, the specific pathogenesis of Mycoplasma pneumoniae has not yet been understood. Recently, researchers have found a protein that called community-acquired respiratory distress syndrome toxin (CARDs TX), which is considered to be the single virulence factor of Mycoplasma pneumonia so far, and played a vital role in the pathogenesis of Mycoplasma pneumoniae. In this research, we attempt to construct the pET28a-CARDs TX recombinant plasmid, observe the expression of it in BL21and purify the pET28a-CARDs protein. Then the purified proteins would be used as antigen to make the antiserum in animals, and the titer and specificity of antiserum would be detected to valuate its application in clinical.Methods:1. Construction and identification of recombinant plasmids: Mycoplasma pneumoniae genome was used as a template, and use PCR to amplify CARDs TX gene (MPN372) that with BamH I and Xhol I restriction sites, clone it into the pET28a plasmid. Obtain the pET28a-CARDs TX recombinant plasmid that can successfully express in BL21after8times point mutation (UGA-UGG). And the recombinant plasmids also would be identified use digesting and sequencing technique.2. Expression and identification of recombinant plasmids:Transform the pET28a-CARDs TX recombinant plasmids into BL21, and then induce the BL21bacteria that have recombinant plasmid to express CARDs TX protein by the use IPTG. Detect the expression and solubility of CARDs TX protein by SDS-PAGE and Wetern Blot method, respectively.3. Purification and identification CARDs TX protein:Purify the CARDs TX protein by use of affinity chromatography (Ni-NTA). And identificate the purification conditions of the protein by SDS-PAGE and Wetern Blot method.4. Preparation and characterization of polyclonal antibodies:Two male rabbits were adaptive feed for2weeks before the first immunization, the lml lmg/ml protein with an equal volume of Freund’s complete adjuvant were thoroughly mixed and immunized subcutaneously into the rabbits.2weeks later, booster immunization every one week multipoint use1ml lmg/ml protein with an equal volume of Freund’s incomplete adjuvant. The ear vein blood of rabbit that pre-immune was collected as control, and the neck arteries blood was collected after two weeks of the last immune. The potency and specificity of the antirera was detected bi the use of Western blot method, and its clinical value was evaluated by ELISA assay.Results:1. The exogenous gene, vector and recombinant plasmid were all in the correct size and position by enzyme digestion identification. The exogenous DNA sequence were consistent with MPN372bases sequence published in Genbank, and mutant all8UGA sites into UGG successfully after sequencing identification.2. We found that the protein at68kDa position has increased significantly after induction of IPTG according to the SDS-PAGE and Wetern Blot results. We also found that the protein is not soluble.3. After the use of affinity chromatography techniche and the purified protein was under Wetern Blot and SDS-PAGE analysis, we found a specific purpose band in the68kDa position.4. Specific and high-titer recombinant polyclonal antibody to CARDs TX has been successfully obtained, and has good application value in the detection of Mycoplasma pneumoniae, and provides the basis for the prevention, diagnosis and treatment of Mycoplasma pneumoniae.Conclusion:The pET28a-CARDs TX recombinant plasmid was constructed successfully. After induction with IPTG, there was a significant increase expression of the CARDs TX protein in BL21. The CARDs TX protein was purificated out successfully and we also successfully prepared high titer and specific polyclonal antibodies.