禽白血病病毒p27蛋白多克隆抗体的制备及POCT检测方法的建立

doi:10.7668/hbnxb.20192997

摘要/Abstract

摘要：

旨在制备禽白血病病毒p27蛋白多克隆抗体和建立一种简便准确的禽白血病病毒的检测方法,通过扩增禽白血病病毒p27目的基因,构建pGEX-6p-1-p27、pET-32a-p27蛋白原核表达载体。将蛋白分别免疫BALB/c小鼠和新西兰白兔,制备鼠源多克隆抗体和兔源多克隆抗体,制备鼠源多克隆抗体偶联荧光微球,利用三维喷点平台将鼠源多克隆抗体IgG喷涂到样品垫;用划膜仪将纯化后的兔源多克隆抗体IgG和山羊抗兔抗体稀释液划到NC膜上,作为T线和C线,进行试纸条的组装,初步建立了即时检验荧光微球免疫层析检测ALV试纸法,并用POCT试纸条检测临床阳性样本。结果表明:成功构建pGEX-6p-1-p27、pET-32a-p27蛋白原核载体并诱导p27重组蛋白表达。将纯化后的p27融合蛋白与佐剂等容量混合后作为免疫原免疫BALB/c小鼠和新西兰白兔,成功制备出p27蛋白的鼠源和兔源多克隆抗体,通过血清效价以及Western Blot鉴定显示多抗的免疫原反应良好。荧光微球偶联鼠源多克隆抗体的最适pH值为6.2。POCT试纸条检测结果显示,ALV的阳性样品与荧光微球包被的鼠源多克隆抗体结合良好,T/C数据比其他样本高,且仅有阳性样本和荧光微球包被的鼠源多克隆抗体结合后,在紫外灯的照射下,C线(质控线)和T线(检测线)才均会有荧光条带,与临床诊断结果相符。试验建立的POCT荧光微球免疫层析检测ALV的方法,可为禽白血病病毒的检测提供一种更为简便的方法,为基层兽医临床检测提供便利。

关键词: ALV, p27, 原核表达, 多克隆抗体, 荧光微球免疫层析

Abstract:

It aimed to develop a polyclonal antibody to Avian leukosis virus (ALV)and to establish a simple and accurate method for the detection of ALV.pGEX-6p-1-p27 and pET-32a-p27 protein vectors were constructed by amplifying the target gene of ALV p27.The proteins were immunized in BALB/c mice and New Zealand white rabbits,and mouse-derived polyclonal antibodies and rabbit-derived polyclonal antibodies were prepared.The mouse-derived polyclonal antibody coupled fluorescent microspheres were prepared,and the mouse-derived polyclonal antibody IgG was sprayed onto the sample pads using a three-dimensional spray point platform;the purified rabbit-derived polyclonal antibody IgG and goat anti-rabbit antibody dilution were scribed onto the NC membrane using a scribing instrument as T and C lines for the assembly of test strips,and a preliminary Point-of-care testing(POCT)immunochromatographic assay was established.The test strips were assembled and a preliminary point-of-care testing(POCT)method was developed for the detection of ALV by fluorescent microsphere immunochromatography.The results showed that the prokaryotic expression vectors for pGEX-6p-1-p27 and pET-32a-p27 were successfully constructed and the expression of p27 recombinant protein was induced.After mixing the purified p27 fusion protein with the adjuvant as an immunogen immune BALB/c mouse and a New Zealand white rabbit,a mouse-derived and rabbit-derived polyclonal antibody of the p27 protein was successfully prepared,and the immunogen response of the multi-antibody was shown to be good by serum potency and Western Blot identification.The optimal pH of the fluorescent microsphere-coupled murine polyclonal antibody was 6.2.POCT strips showed that positive samples for ALV bound well to the fluorescent microsphere-coated murine polyclonal antibody,with higher T/C data than other samples,and that positive samples bound to the fluorescent microsphere-coated polyclonal antibody showed fluorescent bands in the C line(quality control line)and T line(detection line)under UV light,which the results were consistent with clinical diagnostic results.It can be concluded that the POCT fluorescent microsphere immunochromatographic detection method for ALV established provides a simpler method for the detection of Avian leukosis virus and facilitates clinical detection for primary veterinarians.

Key words: Avian leukosis virus, p27, Prokaryotic expression, Polyclonal antibody, Fluorescent microsphere immunochromatography

邵颖, 黄燕, 杨艳, 龚柳菲, 宋祥军, 涂健, 祁克宗. 禽白血病病毒p27蛋白多克隆抗体的制备及POCT检测方法的建立[J]. 华北农学报, 2022, 37(6): 201-209. doi: 10.7668/hbnxb.20192997.

SHAO Ying, HUANG Yan, YANG Yan, GONG Liufei, SONG Xiangjun, TU Jian, QI Kezong. Preparation of Avian leukosis virus p27 Protein Polyclonal Antibody and Estab lishment of POCT Detection Method[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 201-209. doi: 10.7668/hbnxb.20192997.

http://www.hbnxb.net/CN/Y2022/V37/I6/201

图/表 20

图1 pET-32a-p27基因的扩增 M.DL2000 plus Marker;1,2.pET-32a-p27目的片段。

Fig.1 Amplification of pET-32a-p27 gene M.DL2000 plus Marker;1,2.pET-32a-p27 target fragment.

图2 pGEX-6P-1-p27基因的扩增 M.DL2000 plus Marker;1,2.pGEX-6P-1-p27目的片段。

Fig.2 Amplification of pGEX-6P-1-p27 gene M.DL2000 plus Marker;1,2.pGEX-6P-1-p27 target fragment.

图3 pGEX-6P-1-p27重组质粒的构建 M.DL2000 plus Marker;1—3.pGEX-6P-1-p27重组质粒。

Fig.3 Construction of pGEX-6P-1-p27 recombinant plasmid M.DL2000 plus Marker;1—3.pGEX-6P-1-p27 DNA recombinant plasmid.

图4 pET-32a-p27重组质粒的构建 M.DL2000 plus Marker;1—3.pET-32a-p27重组质粒。

Fig.4 Construction of pET-32a-p27 recombinant plasmid M.DL2000 plus Marker;1—3.pET-32a-p27 DNA recombinant plasmid.

图5 His-p27融合蛋白在大肠杆菌中的诱导表达 M.蛋白分子量标准;1.pET-32a空质粒;2.His-p27未诱导;3—5.His-p27全菌蛋白、上清和沉淀。

Fig.5 His-p27 induced expression of recombinant protein in Escherichia coli M.Protein Marker;1.pET-32a empty plasmid;2.His-p27 was not induced;3—5.Whole bacterial protein,supernatant and precipitation after His-p27 induction.

图6 GST-p27融合蛋白在大肠杆菌中的诱导表达 M.蛋白分子量标准;1—3.0.5 mmol/L IPTG,诱导温度分别为16,28,37 ℃的上清蛋白;4—6.0.5 mmol/L IPTG,诱导温度分别为16,28,37 ℃的包涵体;7—9.0.5 mmol/L IPTG,诱导温度分别为16,28,37 ℃的全菌蛋白。

Fig.6 Induced expression of GST-p27 recombinant protein in Escherichia coli M.Protein Marker;1—3. 0.5 mmol/L IPTG with induction temperatures of 16, 28, 37 ℃ supernatant protein, respectively; 4—6. 0.5 mmol/L IPTG and induction temperatures of 16, 28,37 ℃ for inclusion bodies, respectively; 7—9. 0.5 mmol/L IPTG and induction temperatures of 16, 28,37 ℃ for whole bacteriophage protein, respectively.

图7 His-p27融合蛋白纯化 M.蛋白分子量标准;1.上清;2—3.流穿液;4—9.洗脱蛋白。图8同。

Fig.7 His-p27 recombinant protein purification M.Protein Marker weight standard;1.Supernatant;2—3.Protein flow-through fluid;4—9.Eluted protein.The same as Fig.8.

图8 GST-p27融合蛋白的纯化 4—8.洗脱蛋白。

Fig.8 Purification of GST-p27 recombinant protein 4—8.Eluted protein.

表1 鼠源抗原稀释度

Tab.1 Mouse-derived antigen dilution

血清稀释度 Serum dilution		包被抗原稀释度Dilution of coated antigen
血清稀释度 Serum dilution		1∶100	1∶200	1∶400	1∶800	1∶1 600	1∶3 200
1∶100	+	2.53	1.83	1.70	2.33	2.21	2.55
	-	0.06	0.06	0.07	0.06	0.05	0.06
	P/N	41.29	30.53	25.86	40.92	42.18	40.62
1∶200	+	2.17	2.11	2.14	1.95	1.91	2.43
	-	0.06	0.06	0.06	0.05	0.05	0.07
	P/N	38.08	38.07	38.27	38.31	38.65	33.68
1∶400	+	3.04	2.26	2.45	2.62	3.30	3.25
	-	0.05	0.06	0.05	0.06	0.06	0.06
	P/N	56.43	40.89	45.01	46.23	59.62	52.14
1∶800	+	2.14	1.83	2.34	2.20	2.50	2.98
	-	0.08	0.05	0.05	0.06	0.05	0.06
	P/N	28.18	34.22	44.62	37.88	50.32	48.19

表2 鼠源多克隆抗体血清效价

Tab.2 Mouse-derived polyclonal antibody serum titer

血清稀释度 Serum dilution	阳性对照 (P)OD值 Positive control (P) OD value	阴性对照(N) OD值 Negative control (N) OD value	P/N
1∶400	3.715	0.057	65.35
1∶800	3.172	0.047	66.98
1∶1 600	2.780	0.047	59.15
1∶3 200	2.197	0.048	46.19
1∶6 400	2.287	0.045	50.64
1∶12 800	2.105	0.046	45.31
1∶25 600	2.020	0.050	40.19
1∶51 200	1.770	0.048	37.22
1∶102 400	1.527	0.049	30.94
1∶204 800	1.086	0.049	22.27
1∶409 600	0.408	0.048	8.53
1∶819 200	0.272	0.047	5.81
1∶1 638 400	0.055	0.049	1.10

表3 兔源抗原稀释度

Tab.3 Rabbit-derived antigen dilution

血清稀释度 Serum dilution		包被抗原稀释度Dilution of coated antigen
血清稀释度 Serum dilution		1∶100	1∶200	1∶400	1∶800	1∶1 600	1∶3 200
1∶100	+	2.43	2.42	2.48	2.96	2.90	2.36
	-	0.21	0.22	0.23	0.24	0.20	0.19
	P/N	11.73	11.25	10.67	12.35	14.64	12.16
1∶200	+	2.30	2.78	2.73	2.89	3.12	2.29
	-	0.12	0.13	0.13	0.14	0.11	0.12
	P/N	18.53	22.06	21.80	20.39	29.35	19.77
1∶400	+	2.13	2.79	2.60	3.22	3.00	2.88
	-	0.10	0.09	0.09	0.10	0.08	0.08
	P/N	21.13	30.16	27.91	32.82	36.47	33.92
1∶800	+	2.65	2.76	2.63	3.26	3.08	2.01
	-	0.08	0.08	0.09	0.08	0.08	0.07
	P/N	31.32	36.59	30.24	41.36	40.21	27.04

表4 兔源多克隆抗体血清效价

Tab.4 Rabbit-derived polyclonal antibody serum titer

血清稀释度 Serum dilution	阳性对照 (P)OD值 Positive control (P) OD value	阴性对照(N) OD值 Negative control (N) OD value	P/N
1∶400	3.200	0.333	13.10
1∶800	3.371	0.183	18.94
1∶1 600	2.896	0.146	22.95
1∶3 200	3.051	0.086	37.44
1∶6 400	3.249	0.090	36.92
1∶12 800	3.324	0.104	38.69
1∶25 600	2.920	0.127	23.17
1∶51 200	2.792	0.140	23.43
1∶102 400	1.879	0.139	14.36
1∶204 800	0.942	0.097	9.72
1∶409 600	0.529	0.142	4.33
1∶819 200	0.352	0.212	1.95

图9 鼠源多克隆抗体Western Blot 1.pGEX-6P-1;2.GST-p27蛋白。

Fig.9 Western Blot of mouse-derived polyclonal antibody 1.pGEX-6P-1;2.GST-p27 protein.

图10 兔源多克隆抗体Western Blot 1.pET-32a;2.His-p27蛋白。

Fig.10 Western Blot of rabbit-derived polyclonal antibody 1.pET-32a;2.His-p27 protein.

表5 POCT试纸条读数结果

Tab.5 POCT test paper reading results

pH		样本1 Sample 1	样本2 Sample 2	样本3 Sample 3	平均值 Average
6.2	T	2 657 778.00	4 843 399.00	2 028 170.00	3 176 449.00
	C	82 026 386.00	69 707 350.00	147 935 698.00	99 889 811.33
	T/C	0.03	0.07	0.01	0.04
6.6	T	2 335 250.00	2 275 069.00	2 275 069.00	2 295 129.33
	C	68 544 692.00	57 300 177.00	57 300 177.00	61 048 348.67
	T/C	0.03	0.04	0.04	0.04
7.2	T	2 827 893.00	3 428 148.00	1 527 209.00	2 594 416.66
	C	34 929 902.00	62 498 742.00	143 510 954.00	80 313 199.33
	T/C	0.08	0.05	0.01	0.05

图11 阴性结果

Fig.11 Negative result

图12 阳性结果

Fig.12 Positive result

图13 弱阳性结果

Fig.13 Weak positive

表6 POCT试纸条读数结果

Tab.6 POCT test paper reading results

样本 Sample	组别 Group	T T	C C	T/C(面积) T/C(Area)
禽白血阳性样本	A	9 245 925	13 633 183	0.11
Positive avian	B	9 530 030	15 372 915	0.12
White blood	C	9 463 441	13 622 658	0.13
samples	D	9 337 630	12 830 940	0.14
禽腺病毒液	A	8 811 389	12 982 147	0.04
Avian	B	8 571 000	9 952 393	0.01
adenovirus	C	8 869 786	15 358 300	0.03
liquid	D	8 623 897	9 379 924	0.03
PBS	A	8 611 921	10 138 380	0.01
	B	8 626 028	9 187 230	0.01
	C	8 619 161	9 161 213	0.01
	D	8 557 189	9 365 636	0.01

图14 试纸条检测 A.试纸条组装;B.试纸条成品;C.加入样本后紫外灯照射下试纸条结果。

Fig.14 Test paper test A.Test strip assembly;B.Test strip finished product;C.Add sample after ultraviolet lamp irradiation test strip result.

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