| Avian leukemia is a general term for a variety of neoplastic diseases caused by avian leukosis virus(ALV).Avian leukosis virus subgroup A(ALV-A),avian leukosis virus subgroup B(ALV-B)and avian leukosis virus subgroup J(ALV-J)are the three most common subgroups of infecting chickens.At present,flocks eradication is the most effective way to prevent and control ALV.Antibody detection is an important way for ALV detection.This study established A/B subgroup ALV ELISA(enzyme-linked immunosorbent assay)antibody detection and J subgroup ALV ELISA antibody detection to providing technical support for domestic ALV prevention and control.In order to produce the coating protein of the antibody ELISA detection method,firstly,293 F cell lines stably expressing the coating proteins ALV-A,ALV-B,and ALV-J gp85 were established respectively.To constuct lentiviral expression plasmids pLVX-A-gp85,pLVX-B-gp85 and p LVX-J-gp85,the gp85 gene was amplified from the cDNA of RAV-1(ALV-A),RAV-2(ALV-B),and HLJ09MDJ-1(ALV-J)by PCR and cloned into pLVX vector.Lentivirus vector was co-transfected into 293 T cells with psPAX2 and pMD2.G plasmids expressing three subgroups of gp85 lentivirus.The harvested lentiviruses were infected with 293 F cells respectively,and then flow cytometry sorting was conducted.After sorting,the proportion of 293 F cells expressing green fluorescence protein(GFP)positive 293 F cells rate reached more than 90%.The sorted cells were cultured for 5 days,and the results of supernatant protein secretion detected by Western Blot showed that the 293 F cell line could secrete gp85 protein.The stability results indicated that GFP and the ALV-A,ALV-B,ALV-J gp85 protein were stably expressed in the 5th,10 th,and 15 th generations of 293 F stable cell line.It proved the establishment of the 293 F stable cell line was successful.After protein purification,the amount of target protein was 30 mg/L.The gp85 protein of ALV-A and ALV-B expressed in 293 F stable cell lines was used as coated antigen 1:1,and A/B subgroup ALV ELISA antibody detection assay was established.The specificity results showed that ELISA did not react with antibodies against other common avian viruses or with antibodies targeting ALV-J,it is better than the detection of IDEXX.For ALV-A positive serum,the sensitivity of this ELISA(1:6400 diluted in serum)was 4 times than that of indirect immunofluorescence assay(IFA;1:1600 diluted in serum).For ALV-B positive serum,the sensitivity of this ELISA(1:12800 diluted in serum)was 8 times than that of IFA(1:1600 diluted in serum).The coefficients of variation(CV)of the intra-and inter-batch repetitive tests were both less than 10%.The results of 1428 clinical serum tests showed that the coincidence rate of ELISA and IFA was 98.81%.ALV-J gp85 protein expressed in 293 F stable cell lines was used as the coated antigen to establish the J subgroup ALV ELISA antibody detection assay.The specificity results showed that ELISA did not react with antibodies against other common avian viruses or with antibodies targeting ALV-A,ALV-B,it is better than the detection of IDEXX.The sensitivity of this ELISA(1:51200 diluted in serum)was 2 times than that of the detection of IDEXX(1:25600 diluted in serum)and 16 times than that of IFA(1:3200 diluted in serum).The CV of the intra-and inter-batch repetitive tests were both less than 10%.A total of 1180 clinical sera were detected with this ELISA method and IFA,and the coincidence rate of the two was as high as 98.31%.In this study,a 293 F cell line capable of stably expressing ALV-A,ALV-B,and ALV-J gp85 proteins was successfully constructed.Using purified gp85 protein as the coating antigen,an ELISA method for simultaneous detection of ALV-A/B antibodies and an ELISA method for detection of ALV-J antibodies were successfully established.These two methods have strong specificity,high sensitivity,good stability,and high agreement with IFA,which provide important technical means for serological monitoring,diagnosis and eradication of ALV. |
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