| Chicken infectious anemia virus(CIAV)is a pathogen of chicken infectious anemia(CIA),which can cause clinically aplasia bone marrow in young chickens and systemic lymphoid tissue atrophy.CIAV will attack the host's immune system,which will not only cause secondary infections of other pathogens,but also interfere with the immune effect of major diseases.The disease can be passed from hen to offspring,so it is very important to timely detect and eliminate diseased breeders by monitoring antibody of breeders.Foreign commercial ELISA CIAV antibody detection kits are expensive,and the cost of antibody detection for this disease is too high,there are no related commercial detection products for CIAV in China.Therefore,in this study,the CIAV capsid protein VP1 was used as an antigen to establish a CIAV indirect ELISA antibody detection method with specificity,sensitivity,and good repeatability,hoping to provide an important tool for CIAV antibody detection and disease elimination.The main contents are as follows:1.Expression of recombinant VP1 protein by baculovirus expression systemThe transfer plasmid containing the VP1 gene and the baculovirus genome were co-transfected into sf 9 cells to produce a recombinant baculovirus carrying the VP1 gene,which was named BAC-VP1.Under the light microscope,it was observed that the transfected cells became larger and swelled compared to normal cells,and it was initially determined that the recombinant baculovirus was formed.The genome of cells infected with recombinant virus was extracted and amplified by PCR.The PCR product was subjected to nucleic acid electrophoresis and the target band was detected,it was further confirmed that the recombinant baculovirus BAC-VP1 was successfully constructed.After harvesting the cells infected with BAC-VP1,the samples were subjected to Western Blot.No target band was detected,indicating that the recombinant VP1 protein was not successfully expressed.2.Expression of recombinant VP1 protein by prokaryotic expression systemFurthermore,E.coli expression system was used to realize the expression of recombinant VP1 protein.The CIAV gene sequence isolated in our laboratory was optimized for E.coli codons and then sent to a biological company for synthesis.The truncated recombinant VP1(40?449aa)protein and recombinant VP1(78?449aa)protein expression plasmids were respectively transformed into BL21 strain.After induction by IPTG,the results of SDS-PAGE showed that the recombinant VP1(40?449aa)protein and the recombinant VP1(78?449aa)protein were successfully expressed and were mainly insoluble.Subsequently,recombinant VP2 protein and five different molecular chaperones have been tried to help recombinant VP1 protein achieve soluble expression.Finally,the soluble expression of recombinant VP1(40?449aa)protein was successfully realized by using molecular chaperone No.3(dna K-dan J-grp E),and the soluble expression of recombinant VP1(40?449aa)protein was successfully realized by molecular chaperon No.5(dna K-dan J-grp E/gro ES-gro EL-tig).3.Purification of recombinant VP1 protein expressed in prokaryoticThe truncated recombinant VP1(40?449aa)protein and recombinant VP1(78?449aa)protein expressed in prokaryotic cells were purified by affinity chromatography,and the eluted protein samples were analyzed by SDS-PAGE.The results showed that the truncated recombinant VP1(40?449aa)protein did not bind to the Ni-NTA affinity chromatography column,and there was no target protein in the eluted sample,while the recombinant VP1(78?449aa)protein yielded more and the purity was higher under the concentration of 300 mmol/L imidazole.4.Establishment of CIAV indirect ELISA antibody detection method based on recombinant VP1 protein expressed by E.coli BL21 strainThe purified recombinant VP1(78?449aa)protein expressed by E.coli BL21 strain was used as a diagnostic antigen to establish a CIAV indirect ELISA antibody detection method.The optimal reaction conditions of a CIAV indirect ELISA antibody detection method were determined as follow: the concentration of coating antigen was 16 ?g/m L,sample serum was diluted in 1:80,the coated condition was overnight in 4?,the blocking time was 2h in 37?,and the enzyme conjugated antibody was diluted in 1:10 000.30 CIAV negative sera were tested by this method,which determined that the value of cut-off was 0.285 in this method.When using the established CIAV indirect ELISA antibody detection method to detect the pathogenic sera of AIV,NDV,MDV,and IBDV,the results are all negative,indicating that the method has good specificity.Using this method and IDEXX's CIAV ELISA antibody detection kit to simultaneously detect CIAV positive sera of different dilution multiples,the minimum detection dilution of this method is 512-fold dilution,while IDEXX's CIAV ELISA antibody detection kit is 256-fold dilution,indicating that the method has good sensitivity.Repeatability tests show that the coefficients of variation within and between batches are less than 10%.Using this method and IDEXX's CIAV ELISA antibody test kit to simultaneously detect 90 clinical sera and calculating their coincidence rate,the results showed that IDEXX's CIAV ELISA antibody test kit detected 50 positive samples,of which 45 samples were positive by this method and the coincidence rate of positive samples is 90%;IDEXX's CIAV ELISA antibody test kit detected 40 negative samples,of which 32 samples were negative by this method,and the coincidence rate of negative samples is 80%,the overall coincidence rate is 85%. |
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