Preparation And Preliminary Identification Of Antibodies To The EGF And SP Functional Domain From MASP-2

In recent years, the theroy of pattern recognition concerning innate immune cells has been put forward and it promotes the development of immunology. It was paid highly attention to the pattern recognition receptor-pathogen associated molecule pattern(PRR-PAMP). Mannose-binding lectin (MBL) is one of the important PRRs and plays a key role in innate immunity. When MBL recognizes pathogens, mannose-binding lectin associated serine protease-2(MASP-2) as the downstream proteinase of MBL plays an important role in the lectin pathway of complement activation. MASP-2comes up self-activation by decomposing peptide bond of arginine-isoleucine of the N end of serine protease domain. In Addition, MASP-2was related to tumors, infectious diseases and autoimmune diseases, et al. Therefore, the research on MASP-2has become one of the hot topics in innate immunity. In this study, our plan was to construct recombinant prokaryotic expression vector of the EGF and SP functional domain and express the EGF and SP functional protein from MASP-2, and then prepare their polyclonal antibodies for the detection of MASP-2and the analysis of MASP-2expression in different cells.Firstly, the total RNA was extracted from the fetal liver with Oligo(dT) as a primer, and the cDNA of human masp-2was transcripted reversely from total RNA, and then the genes coding for EGF and SP domain were amplified respectively by PCR with cDNA as template. The PCR products were purified through phenol-chloroform extraction, then the EGF, SP fragments and pGEX-6p-2plasmid were digested by BamH I and Not I, and then purified again with gel recovery. Secondly, the digested products of EGF and SP fragments were connected to the digested pGEX-6p-2vector with T4DNA ligase to construct recombinant plasmid respectively. The EGF, SP recombinant plasmids were transformed into E.coli competent BL21respectively by the Heat Shock method, then the transformed bacteria were smeared directly onto LB plate and then cultured overnight. Thirdly, single colony grown on the LB plate was screened by PCR with the universal primers for pGEX-6p-2; then the positive single colony was inoculated in LB liquid selective medium and cultured in the shaker overnight at37℃. Lastly, the plasmids were extracted from the pure culture as template, cross-PCR (with universal primer and specific primer) and digestion method(with BamH I and Not I) were used to identified their correctness preliminarily. The plasmids with both positive results were sequenced to further identify the correct results of recombinant plasmids. The results of DNA Sequence were consistent with masp-2EGF and SP standard sequence from GenBank entirely, and no drift was found. It meant the recombinant pGEX-6P-2-EGF and pGEX-6P-2-SP were successfully constructed.After identification by restrictive enzymes digestion and sequencing, BL21with the recombinant plasmid pGEX-6P-2-EGF, pGEX-6P-2-SP was cultured overnight respectively. The second day, IPTG was added into the enlarged cultivation of bacteria to induce protein expression, then the culture was collected by centrifugation at4℃. The supernatant was disposed of, and the pellet(bacteria) was washed with Tris-HCL buffer, again. After ultrasonic degradation of bacteria, the supernatant was separated from pellet by centrifugation respectively, and both of them were taken a little out as sample for SDS-PAGE, then SDS-PAGE gel was stained with Coomassie brilliant to analyze expression of fusion protein. GST-EGF fusion protein as a soluble protein was expressed in the supernatant was purified from the supernatant; and SP fusion protein existed only in the form of inclusion body; after urea treatment, GST-SP fusion protein was purified.The6-7aged BALB/c mice were immunized with purified GST-EGF and GST-SP fusion protein respectively. Mice were immunized via intraperitoneal injection three times,0,21,35days respectively. Serum was collected in7days after the last injection. The specificity of the polyclonal antibody was assayed with Western blotting, and the titer was determined by indirect ELISA. The titer of EGF antibody was more than1:32000and the titer of SP antibody was more than1:40000. WB analysis showed the serum of fusion protein appeared the specific target bands after1000-fold dilution, the control group did not appear. Strong specificity and higher titer EGF and SP antibody were generated.