Establish Of Immune Turbidimetric Quantitative Detection Method For Canine C-reactive Protein

C-reactive protein is a kind of trace protein in serum, initially named for its capacity to precipitate pneumococcal cell wall polysaccharide. It was the first time to describe the C-reactive protein. CRP is a kind of acute phase response protein, the level is low in normal canine scrum, but it will rapidly increase while the body under the stimulation of tissue injury and the amplification will increase in proportion to the stimulus intensity. CRP index can be used to indentify the bacterical infection and virus infection in dogs, in order to guide the use of antibiotics. CRP is one of the routine testing indicators as white cells count and red cell count in human clinical medicine, but the CRP detection technology has not been used in veterinary clinic. This experiment cloned the canine CRP gene, established the pEASY-El-CRP prokaryotic expression vector, expressed the recombinant canine CRP, and purified the recombinant protein. Affinity chromatography was used to purify the serum canine CRP. The experiment manufactured and purified the polyclonal antibody of rabbit anti-canine serum CRP and rabbit anti-canine fusion CRP, and compared their immunogenicity. We finaly established the immune transmission turbidimetric quantitative detection method with polyclonal antibody of rabbit anti-canine fusion CRP, and it laid the foundation for the use of canine CRP detection technology in veterinary clinic.The experiment clone the canine CRP gene, established the prokaryotic expression vector, purified of the expressed canine fusion CRP. The length of Canine CRP cDNA is672bp. It is made up of223amino acids, the first19form signal peptide,19to20are split shear point of signal peptide with main protein. In all of amino acid,21.97%for alpha helical structure, consists of49amino acids;32.74%for extending chain, composed of73amino acids;4.9%for beta Angle, consists of11amino acids;40.36%for random curl, composed of90amino acids. The amino acid sequence contains an N-glycosylation site which belongs to sugar protein; a casein kinase II phosphorylation sites, two protein kinase C phosphorylation sites, two N-14phthalein base sites and tyrosine kinase phosphorylation site. Comparison the canine CRP gene sequence this experiment obtained with that in NCBI GenBank, we find out that the gene sequence homology is99%. The experiment established the pEASY-El-CRP prokaryotic expression vector, analysised the expressed recombinant protein with SDS-PAGE. There is a band in the maker of25kDa, which is consistent with the prediction of software that is24.64kDa. There is a band in the maker of 25kDa after SDS-PAGE and Western blotting analysis the purified recombinant protein.Subcutaneous injection of turpentine for canine, we obtained the acute phase serum, and purified it with affinity chromatography. There is a band of in the maker of25kDa after SDS-PAGE analysis.The experiment manufactured and purified the rabbit anti-canine serum CRP and rabbit anti-canine fusion CRP, and purified their polyclonal antibodies. Through the SDS-PAGE test we can found that both of them had two bands in the marker of55kDa and25kDa. Western blotting test can find stripe obviously, but the stripe of polyclonal antibodies of recombinant protein is more obviously. Antibody titers test with ELISA, their titers are1:1024and1:2048.We established immune transmission turbidimetric quantitative detection method by using polyclonal antibody of rabbit anti-canine fusion CRP. The reagent that the experiment established stability is good, the intra-and interassay iterancy are good both with methodological evalution test. It has significant difference between the experiment group and the control group, P<0.