The Expression And Purification Of Measles Virus N Protein And Its Preliminary Application In Colloidal Gold Immune Chromatography

Measles is a high infectious respiratory disease, The application rates declinedsignificantly after used live attenuated since the early1960s in our country, but every yearthere are still sending out patients within a certain range, and even cause death. Therefore,improv and rapid diagnosis technology grasps the measles epidemic prevention and control ofthe disease is particularly important.Nucleocapsid (N) is the most abundant protein in measles virus particles, It is the firstsynthesis of proteins in the process of reproduction and transcirption, and has stronger antigenactivity, is one of the main antigen of measles vims. This expeirment according to measlesvirus N protein released on NCBI gene sequence, using the premerS.O design pirmers. Fromplasmid containing all measles antigen gene sequences, get purpose gene fragments by PCRamplification, and carirer pET-32a (+) separately BamR I and Xho I double enzyme,T4DNA connection, then connect the product to competent escherichia coli cells e.c. with ourfabrication: oli BL21(DE)3, pick the single colonies after training for the night, add IPTG incultures inducers induced expression, expression products with sds-page electrophoresisdetection, and to express bacteirum genome sequencing, managed to construct recombinantplasmid, named pET-32a (+)/N. After optimize the induced condition, to determine the bestN protein induced condition is: temperature of37°C, IPTG concentration tendency for0.20mmol/L, the induction time of4h. Target protein (His) tags, facilitate nickel ion affinitychromatography purification, results showed that the protein purity can reach90%. N protein expression was applied to immune chromatography of the colloidal gold teststirp, using sodium citrate reduction method, preparation of the three different particle size ofcolloidal gold solution, through the experiment selected grain diameter uniformity (24nm),stable properties of colloidal gold, and use it to mark the measles virus antigen of N (tag26mu g/mL) preparation of immune colloidal gold, with the purified protein (1.25mg/mL) andN (N protein (1.0mg/mL) package is on the nitrocellulose (NC) membrane respectively as thetest line (T) and quality control line (C), the assembly test, verifies the specificity, sensitivity,repeatability and stability. The expeirmental results show that the stirp has good repeatabilityand stability, and preliminary has reached the requirement of detecting N antibodies ofmeasles vims.