The Prokaryotic Expression Of VP-60 Truncated Gene Of Rabbit Hemorrhagic Disease Virus And The Preliminary Study On The Detection Of Antibody Colloidal Gold Test Paper

Rabbit haemorrhagic disease(RHD)is an acute and highly infectious disease caused by the Rabbit haemorrhagic disease virus whose symptoms includes edema and develop ment of blood clot formation in major organs, haemorrhage in respiratory symptom and e tc. Once the rabbits are infected, the morbidity and mortality of RHD could get as high as 100% among its population. Therefore, RHD is one of the most important diseases that a ffect the rabbit cultivation industry.In this study, we designed the primers forward and reverse primers aimed at 900 bas e pairs upstream (AB region) of gene VP60 according to the published RHDV gene sequ ence. The expression recombination plasmid pCold TF-VP60 was constructed using real-time PCR, enzyme digestion and ligation, and then transformed to competent cell BL21( DE3). Positive clones were screened at 37C in the presence of inducer isopropyl-(3-D-gal actopyranoside (IPTG) (lmol/L)to induce expression and optimize the condition. Then S DS-PAGE analysis and western blot confirmation were confucted on these collected posit ive clones. Finally the recombinant proteins were purified by Ni-TED affinity chromato graphy, and then analyzed by enzyme-linked immuno assay. After 3-4 hours incubation in lmol/L IPTG at 37C,85.3KD proteins were obtained from the expression recombinati on TF-VP60 plasmids. The ELISA analysis results of these showed the expression of the recombinant proteins can specifically bind with the immune positive RHDV antibody wit h high reactivity.The T line and C line are labeled with Goat anti rabbit IgG(H+L) and mouse anti recombinant protein respectively. Then RHDV antibody detection test strips are designed in accordance with them. Through the preparation of collaurum,the marker of recombinant VP60 protein,the preparation of rat recombinant VP60 antibody,the optimization of the protein content of T line and C line,and the sensitivity and specificity test,immuno collaurum test strips which can be used to test rabbit anti RHDV antibody are made preliminary.And this collaurum test strips have good. sensitivity and specificity,whose sensitivity can reach the level of immune sera of 1:16.In addition to the detection line of Rabbit anti RHDV serum is out of red band,while the other negative serum,Rabbit anti Pasteurella multocida serum and PBS all have no color stripe belt,which provides a new testing technique for the prevention and control of RHDV.