Development And Application Of Real-time PCR Method For Detection Of Recombinant Fowlpox Virus RFPV-F-VPO

Taking virus as a vector to prepare the vaccine live vector vaccine is currently one of the main research trends. Fowl pox virus is a common pox viral vector and recombinant vaccinia virus vector still maintains the characteristics of wild-type vaccinia virus. As a result of it, it can infects all the cells from mammalian and express foreign proteins with high expression efficiency compared with other expression vectors.Therefore, it can be used to develop genetic engineering live vector vaccines of poultry and taken as a non-replicating viral vector to develop genetic engineering live vector vaccines of mammals which indicate that there are long-term development prospects for the prevention of diseases in animals besides poultry and humans. Fowl pox virus is a common expression vector and recombinant fowl pox virus vector can express a variety of virus protective antigen gene stably by means of genetic engineering. Recombinant fowl pox virus vector can be inserted different foreign genes to express by their own without interferences from each other which can be used to construct more multivalent vaccines and combined vaccine. Because of its large capacity of vector and efficiency expression,,it gets attention from more and more researchers.By gene transfer technology, the integration of foreign DNA into the host cell chromosome is the premise about the foreign proteins's stable expression as the integration efficiency of foreign gene influences the expression of recombinant protein.The methods about traditional tests and analysis about the level of foreign expression in tissues or cells can not keep pace with the rapid development of research needs. Real-time quantitative PCR technology has developed rapidly in recent years as a detection method. The PCR amplification and product analysis of the whole process are sealed in a single tube which achieve the real-time dynamic detection of amplification products and automatic analysis of data, avoiding the after processing of the amplified products.Besides,it effectively avoids the problem of laboratory cross-contamination. During a short period the level of expression of foreign gene can be determined precisely.Passage the recombinant fowl pox virus rFPV-F-VPO in CEF cells 20 times, extract the virus genome of 1,5,10,15,20 passages and use the fluorescence quantitative PCR reaction conditions after optimization. design and synthesize the specific primers according rFPV-F-VPO foreign gene sequence,at last, make the preparation of standard DNA template. By means of fluorescent dye SYBR Green I, using standard calibration to make standard curve, calculating quantitative virus passages copy number according to the standard curve and then analyzing the expression of the recombinant fowlpox virus containing foreign gene of different cell passages in chicken embryo fibroblasts. Finally, inoculating recombinant fowlpox virus rFPV-F-VPO into SPF chickens,taking of samples the heart, liver, spleen, lung and kidney tissues and organs at different time,using the established real-time PCR method for detection of dynamic distribution of recombinant chicken chicken pox virus live vaccine in body, clarifying the immune mechanism of recombinant fowlpox virus and test data of further research in the clinical application.The results showed that the standard DNA template shows good reproducibility, specificity, and its high sensitivity. In the range of dilution linear concentration, with the reduction of the template, the corresponding Ct increases accordingly. There is linear correlation in them and the r2=0.998, amplification efficiency E= 97.2%. The melting curve is a single peak without non-specific amplification and the Tm melting temperature values are displayed 84±1℃. The analysis of the quantitative virus passages copy number according to standard curve shows that the recombinant fowlpox virus rFPV-F-VPO and VPO gene, F gene, in each generation the level of sub-cells were not the same and expression levels at different times and different organs were not the same situation in chicken.