Development Of Real-time PCR Assay For Detecting RVFV And Expression Of G2 Partial Protein By E.coli

Rift Valley fever virus (RVFV) is a member of Phlebovirus, Bunyaviridae, and it is the causative agent of Rift Valley fever (RVF), which is a peracute or acute zoonotic disease of domestic ruminants. RVF can cause abortions in pregnant animals and a high mortality rate in the newborn animals, and is one of the list A diseases of OIE. This disease is mainly transmitted through mosquitoes. Although the disease has not been found in our country, so far the infected area is expanding in recently years, threatening to our country. It has no research reports targeting to this disease in our country still now. So, it is necessary to establish exact and rapid diagnostic methods for this disease and supervise its activity. In this paper, we described the development of two Real-time PCR assays for detection of RVFV and expression of G2 partial protein by E.coli.Based on the sequences of RVFV G2 gene, two primers and one TaqMan probe were designed in its conservation region. The specificity of primers and TaqMan probe were identified using Blast and PCR. Target sequence was amplified by conventional PCR from plasmid template. After purification, the amplicons was inserted into pMD18-T vector to generate plasmid pMDIS- RG2, which was purified and diluted as the standard templates in the Real-time PCR. After optimizing the PCR conditions, the optimal annealing temperature was 60 C, and Mg2+ concentration was 4mM. The detection limit of this assay was determined at the DNA level of 3 101 copy template. This assay was linear within 61og10 dynamic range, and had a good repeatability.After analysing the G2 gene of the RVFV, one pair of primers was designed for Real-time PCR using SYBR Green I dye. The recombinant plasmid pLSEG-RVFV was used as the template for Real-time PCR after its concentration and purification wasdetermined. The specificity of the primers were identified using Blast and PCR, and the product of Real-time PCR was identified using Melt curve analysis. After optimizing the reaction conditions of Real-time PCR, the optimal annealing temperature was 60 C, and Mg2+ concentration was 3.5mM. The range of this assay could be obtained from 3 106 to 3 102 copy per reaction with a good dynamic range and repeatability.In addition, the G2 protein with highly antigenic portion was selected to be expresed by analysis its amino acid sequence. The interest segment was obtained by PCR, and cloned into plasmid pET32a to generate the plasmid pET32a-RG2p which was screened by colony PCR and restriction enzyme digestion. Its sequence was determined. The recombinant plasmid was transformed into the host strain BL21 (DE3). Expression of target protein was inducted by IPTG, and subsequently analysed by SDS-PAGE and Western-blot. The results showed that target protein was expressed correctly. The production rate of target protein was not improved through optimization of the culture temperature, IPTG concentration and induction time. Therefore, i much work is needed to optimize other conditions to increase the products of target protein.