Preparation And Identification Of Polyclonal Antibodies Against CUB1 And CUB2 Functional Domain From Mannan-binding Lectin Associated Serine Protease-2

As the crucial constituent part of innate immunity, complement is a protein reaction system which has accurate regulatory mechanism.Mannan-binding lectin associated serine protease-2(MASP-2) plays a key role to active the mannose-binding lectin(MBL) pathway that has distinctly important significance of physiology. The deficiency or disfunction of MASP-2 may cause chronic infection. Also it is involved in pathological process of tumors and autoimmune diseases. To measure the concentration of MASP-2 in serum could be an index which will have been used among clinical diagnosis, treatment and research. However, there is no commercial reagent or kid of MASP-2 domestically. The research of MASP-2 mostly focus on complete fragment, but seems not analyze into subfragment. It is necessary to establish a method that could measure the concentrate of MASP-2 accurately and rapidly.The activation occurs after MBL recognize and combine with the pathogen in vivo. MASP-2 as a proteinase located in downstream of MBL occurs self-activation by splitting peptide bond of arginine-isoleucine of the N-end of serine protease domain, casades the response afterwards. MASP-2is composed of 6 function domains. CUB domain is named afrer C1r/ C1 s,Uegf and b?o?n?e? ?m?o?r?p?h?o?g?e?n?e?t?i?c protein-1 where it has been found. CUB1-EGF domain is linked to form homodimer of MASP-2. CUB1-EGF-CUB2 domain is concerned with combination of MBL, and decides that whether it could be activated or not.The design of experiment was about to express the fusion protein CUB1 and CUB2 by constucted recombinant prokaryotic expression vector.Immune the BALB/c mouse about 5 weeks after purify the fusion proteins to preparate the polyclonal antibodies against the CUB1 and CUB2 functional domains.In the first place the primers of CUB1 and CUB2 fragments were designed to be amplified by PCR respectively. The CUB1, CUB2 fragments and pGEX-6p-2 plasmid were digested by BamH I and Not I purified with gel recovery one step further. The fragments were ligated by DNA ligase to complete the construction of recombinant plasmids. The transformed bacteria were smeared directly to LB plate containing Amp(100 μg·mL-1) after the recombinant plasmids were transformed into E.coli competent BL21. The positive single colony was indentified bycross-PCR and digestion method with BamH I and Not I. The fragments were sent to sequence as the final identification.The pGEX-6P-2 prokaryotic vector carrying the target gene CUB1 and CUB2 proliferate till OD reach to 0.8~1.0. IPTG was added into the enlarged cultivation of bacteria to induce protein expression for 3hours.Centrifugated at 4℃ to collect the bacteria. After discarded the supernatant,washed the bacteria with Tris-HCL buffer. After splitted bacteria by ultrasonic, high concertrate urea would be used to dissolve the inclusion body.When the renaturation finished, these two fusion proteins which were purified with Glutathione SepharoseTM4 B beads would be combined with Freund’s adjuvant as antigens to immunize the BALB/c female mouses aged 5 weeks for generating polyclonal antibodies. Subsequently, the specificity of polyclonal antibodies was analysed by Western blot, and the titer was determined by indirect ELISA. Results The fusion proteins GST-CUB1 and GST-CUB2 were successfully expressed and purified.After immunizing BALB/c mouses with these two proteins, thereof, the polyclonal antibodies have been gained. The polyclonal antibodies have high specificity, and there was no cross reaction with other proteins. The titer of anti-CUB1 antibody was more than 1:32 000 and anti-CUB2 antibody was more than 1:16 000, which were determined by indirect ELISA. The polyclonal antibodies aganist GST-CUB1 and GST-CUB2 fusion proteins were obtained respectively with high titer and strong specificity.