Preparation of Polyclonal Antibody Against Recombinant Double CP of Potato leafroll virus and Potato virus S

doi:10.7668/hbnxb.20193055

Abstract

Abstract:

The purpose was to prepare polyclonal antibody against the recombinant double CP of Potato leafroll virus (PLRV)and Potato virus S (PVS),and apply the polyclonal antibody to indirect ELISA and DAS-ELISA detections of PLRV and PVS.Prokaryotic expression vector pET22b-LRCP/SCP of the fused double CP gene of PLRV and PVS was constructed.After replacement of lysozyme treatment by ultrasonic disruption,inclusion body protein was extracted from the recombinant strain BL21(pET22b-LRCP/SCP),the target protein(recombinant double CP)was purified by nickel ion affinity chromatography and high-purity target protein of 51.2 ku was obtained.The high-purity recombinant double CP was used as antigen to immunize rabbits to prepare an antiserum with a titer of 1∶128 k.Specific reactions were respectively observed between the purified polyclonal antibody (IgG) against the recombinant double CP and the positive standard of PLRV or PVS and no cross-reaction was found between the purified IgG and other four potato major viruses (PVX, PVY, PVA and PVM). The purified IgG against the recombinant CP with the diluted concentration of 1∶3 200 still positively reacted with PLRV or PVS in indirect ELISA detection.The purified IgG and the alkaline phosphatase-conjugated IgG both with the diluted contraction of 1∶100 also positively reacted with PLRV or PVS positive standard in DAS-ELISA detection.The results showed that one type of the prepared IgG against the recombinant double CP could detect two viruses of PLRV and PVS by DAS-ELISA or indirect ELISA.

Key words: Potato leafroll virus, Potato virus S, Recombinant double CP, Polyclonal antibody, Indirect ELISA, DAS-ELISA

JI Xiang, SONG Zhicheng, WEI Xiaoling, YANG Yu, SUI Jiongming, GUO Baotai. Preparation of Polyclonal Antibody Against Recombinant Double CP of Potato leafroll virus and Potato virus S[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(4): 212-219. doi: 10.7668/hbnxb.20193055.

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Figures/Tables 10

Fig.1 Restriction identification of prokaryotic expression vector pET22b-LRCP/SCP M.DNA Marker 15000;1.Expression vector pET22b-LRCP/SCP;2.Plasmid pET22b-LRCP/SCP+Hind Ⅲ(+);3-4.Plasmid pET22b-LRCP/SCP+Hind Ⅲ+XhoⅠ.

Fig.2 Expression induction,lysozyme lysis extraction and purification of recombinant double CP of PLRV and PVS M.Protein Marker;1.Uninduced bacterial cells;2.Bacterial cells induced with IPTG;3.Inclusion body;4.Purified recombinant double CP.

Fig.3 Expression induction,ultrasonic disruption extraction and purification of recombinant double CP M.Protein Marker;1.Uninduced bacterial cell;2.Bacterial cell induced with IPTG;3.Dissolved inclusion body protein;4-5.Purified recombinant double CP.

Fig.4 Isolation and purification of IgG against recombinant double CP M.Protein Marker;1.Antiserum against recombinant double CP; 2.Isolated IgG;3.Purified IgG.

Tab.1 Activity determination of IgG against double CP of PLRV and PVS by indirect ELISA(PLRV positive standard,

O D ¯ 450

)

空白对照 Blank control	重组双CP抗体(IgG)的稀释度 Diluted concentration of IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶200	1∶400	1∶800	1∶1 600	1∶3 200	1∶6 400	1∶12 800	阴性对照 Negative control
0.071	0.734	0.679	0.626	0.529	0.439	0.300	0.176	0.121

Tab.1 Activity determination of IgG against double CP of PLRV and PVS by indirect ELISA(PLRV positive standard,

O D ¯ 450

)

空白对照 Blank control	重组双CP抗体(IgG)的稀释度 Diluted concentration of IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶200	1∶400	1∶800	1∶1 600	1∶3 200	1∶6 400	1∶12 800	阴性对照 Negative control
0.071	0.734	0.679	0.626	0.529	0.439	0.300	0.176	0.121

Tab.2 Activity determination of IgG against double CP of PLRV and PVS by indirect ELISA(PVS positive standard,

O D ¯ 450

)

空白对照 Blank control	重组双CP抗体(IgG)的稀释度 Diluted concentration of IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶200	1∶400	1∶800	1∶1 600	1∶ 3 200	1∶ 6 400	1∶12 800	阴性对照 Negative control
0.071	0.632	0.583	0.497	0.329	0.270	0.206	0.174	0.093

Tab.2 Activity determination of IgG against double CP of PLRV and PVS by indirect ELISA(PVS positive standard,

O D ¯ 450

)

空白对照 Blank control	重组双CP抗体(IgG)的稀释度 Diluted concentration of IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶200	1∶400	1∶800	1∶1 600	1∶ 3 200	1∶ 6 400	1∶12 800	阴性对照 Negative control
0.071	0.632	0.583	0.497	0.329	0.270	0.206	0.174	0.093

Fig.5 Specificity detection of IgG against double CP by indirect ELISA

Tab.3 Activity determination AP-conjugated IgG of double CP of PLRV and PVS by direct ELISA(PLRV positive standard,

O D ¯ 405

)

空白对照 Blank control	重组双CP抗体(IgG)AP标记物的稀释度 Diluted concentration of AP-conjugated IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶ 200	1∶ 400	1∶ 800	1∶ 1 600	1∶ 3 200	1∶ 6 400	1∶ 12 800	阴性对照 Negative control
0.077	0.500	0.387	0.268	0.183	0.145	0.120	0.108	0.091

Tab.3 Activity determination AP-conjugated IgG of double CP of PLRV and PVS by direct ELISA(PLRV positive standard,

O D ¯ 405

)

空白对照 Blank control	重组双CP抗体(IgG)AP标记物的稀释度 Diluted concentration of AP-conjugated IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶ 200	1∶ 400	1∶ 800	1∶ 1 600	1∶ 3 200	1∶ 6 400	1∶ 12 800	阴性对照 Negative control
0.077	0.500	0.387	0.268	0.183	0.145	0.120	0.108	0.091

Tab.4 Activity determination of AP-conjugated IgG against double CP of PLRV and PVS by direct ELISA(PVS positive standard,

O D ¯ 405

)

空白对照 Blank control	重组双CP抗体(IgG)AP标记物的稀释度 Diluted concentration of AP-conjugated IgG against double CP							阴性对照 Negative control
空白对照 Blank control	1∶ 200	1∶ 400	1∶ 800	1∶ 1 600	1∶ 3 200	1∶ 6 400	1∶ 12 800	阴性对照 Negative control
0.071	0.555	0.430	0.285	0.202	0.152	0.124	0.117	0.098

Tab.4 Activity determination of AP-conjugated IgG against double CP of PLRV and PVS by direct ELISA(PVS positive standard,

O D ¯ 405

)

空白对照 Blank control	重组双CP抗体(IgG)AP标记物的稀释度 Diluted concentration of AP-conjugated IgG against double CP							阴性对照 Negative control
空白对照 Blank control	1∶ 200	1∶ 400	1∶ 800	1∶ 1 600	1∶ 3 200	1∶ 6 400	1∶ 12 800	阴性对照 Negative control
0.071	0.555	0.430	0.285	0.202	0.152	0.124	0.117	0.098

Tab.5 DAS-ELISA detection of PLRV and PVS positive standards(

O D ¯ 405

)

重组双CP抗体 IgG against double CP	空白对照 Blank control	阴性对照 Negative control	抗原类型Antigen type
重组双CP抗体 IgG against double CP	空白对照 Blank control	阴性对照 Negative control	双重组CP Recombinant double CP	PVS阳性标准物 PVS positive standard	PLRV阳性标准物 PLRV positive standard
IgG(1∶100)	0.082	0.096	0.490	0.262	0.271
IgG(1∶200)	0.078	0.089	0.433	0.207	0.203
IgG(1∶300)	0.081	0.091	0.395	0.166	0.166

Tab.5 DAS-ELISA detection of PLRV and PVS positive standards(

O D ¯ 405

)

重组双CP抗体 IgG against double CP	空白对照 Blank control	阴性对照 Negative control	抗原类型Antigen type
重组双CP抗体 IgG against double CP	空白对照 Blank control	阴性对照 Negative control	双重组CP Recombinant double CP	PVS阳性标准物 PVS positive standard	PLRV阳性标准物 PLRV positive standard
IgG(1∶100)	0.082	0.096	0.490	0.262	0.271
IgG(1∶200)	0.078	0.089	0.433	0.207	0.203
IgG(1∶300)	0.081	0.091	0.395	0.166	0.166

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