猪轮状病毒重组VP6*蛋白的原核表达及间接ELISA检测方法的建立

doi:10.11843/j.issn.0366-6964.2024.09.026

摘要/Abstract

摘要：

旨在建立猪轮状病毒(porcine rotavirus, PoRV)的抗体检测方法。本研究以PoRV截短的VP6*(aa149-332)蛋白作为检测抗原，建立PoRV抗体的间接ELISA检测方法。结果显示：截短的重组VP6*(aa149-332)以包涵体和可溶性两种形式存在，大小为22.5 ku。优化后的ELISA反应条件：VP6*(aa149-332)包被量为25 ng·孔^-1，抗原4 ℃过夜包被，5%脱脂奶粉溶液37 ℃封闭2 h，待检血清1 ∶400稀释、37 ℃孵育30 min，酶标羊抗鼠IgG二抗1 ∶24 000稀释、37 ℃孵育30 min，显色15 min。试验确定阴阳性临界值OD_{450 nm}=0.418，可检测到抗体的最大稀释度为1 ∶3 200，批内和批间重复变异系数均小于10%，并具有较好的特异性。28份血清与Western blot结果符合率为96.42%。ELISA OD_{450 nm}值与中和抗体效价log2相关系数R²=0.878 5。综上所述，本研究建立的PoRV抗体间接ELISA具有较高的特异性和敏感性，可应用于PoRV的临床血清样本抗体检测。

关键词: 猪轮状病毒, VP6*蛋白, 间接ELISA, 原核表达

Abstract:

This study was designed to establish an antibody detection method for porcine rotavirus (PoRV). We utilized the truncated VP6* (aa149-332) protein of PoRV as the detection antigen to develop an indirect ELISA detection method for PoRV antibodies. The truncated recombinant VP6* (aa149-332) exists in two forms: inclusion bodies and soluble, with a size of 22.5 ku. The optimized reaction conditions of the ELISA were as follows: the optimal coating antigen concentration, coating time and temperature were found to be 25 ng per well, and left overnight at 4 ℃; the plates were blocked with 5% skimmed milk at 37 ℃ for 2 hours; the optimal dilution of test serum and the secondary antibody were found to be 1 ∶400 and 1 ∶24 000, respectively. The optimal incubation time and temperature for test serum and the secondary antibody were 30 minutes and 37 ℃, respectively, and the color development time was 15 minutes. Testing 30 negative sera determined the cutoff value at OD_{450 nm}=0.418, the maximum dilution of detectable antibodies is 1 ∶3 200. The intra-batch and inter-batch coefficient of variation were both less than 10%, and it exhibited good specificity. The agreement rate with Western Blot results for 28 sera was 96.42%. The correlation coefficient (R²) between ELISA OD_{450 nm} values and neutralizing antibody log2 values was 0.878 5. In summary, the indirect ELISA established in this study for detecting porcine rotavirus antibodies has high specificity and sensitivity, making it applicable for clinical sample testing of porcine rotavirus.

Key words: porcine rotavirus, VP6* protein, indirect ELISA, prokaryotic expression

中图分类号:

高力国, 申翰钦, 陈诒全, 陈胜, 蔺文成, 陈峰. 猪轮状病毒重组VP6*蛋白的原核表达及间接ELISA检测方法的建立[J]. 畜牧兽医学报, 2024, 55(9): 4021-4028.

Liguo GAO, Hanqin SHEN, Yiquan CHEN, Sheng CHEN, Wencheng LIN, Feng CHEN. Prokaryotic Expression of Recombinant VP6* Protein of Porcine Rotavirus and Establishment of Indirect ELISA Detection Method[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4021-4028.

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	阴性对照 Negative control	PoRV	PEDV	TGEV	PDCoV	PRRSV
OD_{450 nm}	0.130	1.382	0.227	0.292	0.215	0.293
结果 Results	-	+	-	-	-	-

方法 Method		Western blot		合计 Total	符合率/% Coincidence rate
方法 Method		阳性血清 Positive sera	阴性血清 Negative sera	合计 Total	符合率/% Coincidence rate
本试验建立的ELISA检测方法	阳性血清 Positive sera	13	1	14	92.86
The method established in this experiment	阴性血清 Negative sera	0	14	14	100
合计 Total		13	15	28	96.42

猪类别 Pig category	阳性血清 Positive sera	阴性血清 Negative sera	合计 Total	阳性率/% Positive rate
待产母猪 Expectant sow	58	30	88	65.90
断奶仔猪 Weaned piglets	25	67	92	27.17
合计 Total	83	97	180	46.11

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