Expression And Preliminary Identification Of Recombination Protein Mannose-binding Lectin Associated Protein 19

Complement is an important part of the innate immunity, which can be activated by classical pathway, lectins pathway and alternatives pathway. The compound composed of mannan-binding lection(MBL)/ficolin(FCN)and mannose-binding lectin associated serine protease(MASP) is the first complement component to activate lectin pathway. MASP family is composed of MASP-1, MASP-2, MASP-3 serine protease and MAp19, MAp44 non-enzymatic proteins, wherein MASP-2 plays an important role in the lectin pathway activation. MAp19 is one of two proteins arising by alternative splicing of the primary transcript of the MASP-2 gene, which consists of only the CUB1 domain and the EGF domain with an additional small C-terminal tail of four unique acid residues(EQSL). MAp19 can combine with the MBL, but without a catalytic function, whether MAp19 acts as a competitive inhibitor of MASP-2-mediated complement activaton is still to be further study. Beside this, some studies suggest that there are some correlations among MAp19 with kidney disease, myeloma, SARS, etc. In this study, our plan is to construct eukaryotic expression vector pc DNA3.1/Myc-HisA-MAp19 and express MAp19 protein for the further research.Firstly, MAp19 gene was amplified by PCR. The PCR products were purified through gel extraction. Both the purified PCR products and the eukaryotic expression vector pcDNA3.1/Myc-His A were digested by BamHⅠand KpnⅠ, and then purified with gel recovery. Both of the desigsted fragments MAp19 and pcDNA3.1/Myc-HisA were ligated with T4 DNA ligase to construct recombinant plasmid. Secondly, the MAp19 recombinant eukaryotic plasmid was transformed into E.coli BL21 by the Heat Shock method, then the transformed bacteria were smeared directly to LB plate containing Amp, and the cultured overnight. Thirdly, single colony grown on the LB plate was screened by cross-PCR with universal primers for pcDNA3.1/Myc-HisA and specific primers for MAp19, and the digestion method with BamHⅠand KpnⅠwas used to identified its correctness preliminarily. The MAp19 recombinant eukaryotic plasmid was identified by restriction mapping and sequencing. The result of DNA sequence was consistent with MAp19 standard sequence from Genbank entirely, and no frame-shift mutation was found. It meant the recombinant eukaryotic plasmid pcDNA3.1/Myc-HisA-MAp19 was successfully constructed.The recombinant expression vector was transfected into HeLa cells by liposome-mediated method. After forty-eight hours, cells were fixed, blocked and incubated with mouse anti-c-Myc antibody and goat antibody to mouse IgG labelled with Alexa Flour 488. Expression of the MAp19 in transfected HeLa cells was identified by fluorescence microscopy. Different concentrations of G418 were set to determine the minimal lethal does of G418 to the HeLa cells. HeLa cells were cultured selectively under the given concentration of G418. In order to obtain the cell clones possessing plasmid pcDNA3.1/Myc-HisA-MAp19, the He La cells with strong fluorescence were selected to undergo limiting dilution, screening and clone culture. After cultured in large scale, the cells were collected and lysed with protein lysis buffer. Western blot was used to analyze expression of fusion protein Myc-MAp19. After absorpted with anti-c-Myc agarose, the cell lysate and culture supernatant were incubated with mouse anti-c-Myc antibody and goat antibody to mouse IgG labelled with HRP. The Western blot showed that there were two straps with molecular weight of 19.84 KDa, which were accordanced with the size of fusion protein c-Myc-HisA-MAp19. This indicated the fusion protein c-Myc-MAp19 was expressed successfully.