Prokaryotic Expression Of Novel Goose Parvovirus VP3 Protein And Development Of Fluorescent Quantitative PCR Detection Method

Novel goose parvovirus(NGPV)is a Novel agent that causes beak atrophy and dwarfism syndrome(BADS)in ducks,causing Muscovy duck to have a large tongue,short beak,slow growth or dwarfism.Due to the atrophy of beak and stiff tongue,the diseased duck has the eating disorder,diarrhea is thin and the spirit is weak.The disease is characterized by short,weak legs,difficulty in standing and walking,frequent fractures and slow growth.NGPV mostly leads to infection of most meat ducks aged 10-30 d.Although mortality and morbidity are relatively low,it leads to elimination rate of up to 60% of the disabled ducks and stiff ducks in China duck breeding industry,and great economic loss of domestic duck industry.The VP3 protein of the new goose parvovirus contains the main antigen-determining clusters of the virus,which constitutes the main epitope of the virus,and plays an important role in the process of virus adsorption and virus invasion into host cells.Prokaryotic expression vector pCold-TF and transformed into E.coli BL21 competent cells.The VP3 gene expression vector pcold-vp3 was successfully constructed to efficiently express the recombinant protein VP3.High purity recombinant protein VP3 was obtained by His label purification.The protein was present in a soluble form in E.coli BL21 cells.VP3 protein molecular size of 110 kda,Western blot and indirect immunofluorescence identification results showed that the recombinant protein VP3 could react specifically with the monoclonal antibodies of novel goose parvovirus,indicating that the prokaryotic expression recombinant protein VP3 had good immune reactivity,so as to screen out good immune effect single 1-5 and 1-7,the two monoclonal antibodies can be efficiently identified NGPV,for later development of ELISA diagnostic reagents to detect NGPV lay the good foundation.According to the VP3 gene sequence in the NCBI gene library,the internal fragment of VP3 gene was amplified by PCR,and the VP3 gene fragment with molecular size of 143 bp was obtained.Using pmd19-t plasmid as the vector,the recombinant plasmid pMD19-143 was successfully constructed.The positive plasmid was used as a template to establish the real-time fluorescence quantitative PCR standard curve,and the specificity test,sensitivity test and repeatability test were performed.The results show that the fluorescence quantitative reaction procedures: 95? modified 30 s,95? modified 5 s,60? extends 34 s,40 cycles,upstream and downstream primer concentration of 0.4 ?mol/L,the concentration of the probe is 0.8 ?mol/L,the fluorescence quantitative effect is best.The linear correlation coefficient of the standard curve of fluorescence quantitative PCR of NGPV established in this study was all above 0.990.The sensitivity reached 37.2 copies/L,100 times higher than that of conventional PCR.The variation coefficient of intra-batch and inter-batch repeatability was less than 2.2%,indicating good repeatability.Therefore,the fluorescence quantitative PCR method was significantly more sensitive than the conventional PCR method to detect the novel goose parvovirus.This method has the characteristics of simple operation,high sensitivity and strong specificity,and can be initially used for the rapid diagnosis of NGPV epidemic situation.