山羊地方性鼻内肿瘤病毒SU蛋白的表达及其多克隆抗体的制备和特性分析

江锦秀; 张靖鹏; 林裕胜; 游伟; 胡奇林

doi:10.19303/j.issn.1008-0384.2021.02.001

山羊地方性鼻内肿瘤病毒SU蛋白的表达及其多克隆抗体的制备和特性分析

doi: 10.19303/j.issn.1008-0384.2021.02.001

福建省农业科学院畜牧兽医研究所，福建福州 350013

基金项目: 福建省科技重大专项（2019NZ09007）；福建省科技计划公益类项目（2018R1023-1）

详细信息

作者简介:
江锦秀（1987−），女，硕士，助理研究员，主要从事动物传染病研究（E-mail：59714800@qq.com）

通讯作者:
胡奇林（1963−），男，硕士，研究员，主要从事动物传染病学和免疫学研究（E-mail：hql562713@163.com）

中图分类号: S 855.3
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出版历程
- 收稿日期: 2020-09-03
- 修回日期: 2020-12-16
- 网络出版日期: 2021-02-08
- 刊出日期: 2021-02-28

Expression of SU protein of ENTV-2FJ strain, preparation and characterstic analylsis of its polyclonal antibody

Institute of Animal Husbandry & Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujan 350013,China

摘要

摘要: 目的明确山羊地方性鼻内肿瘤病毒（ENTV-2）SU蛋白的功能。方法采用RT-PCR 方法从 ENTV-2FJ分离株中扩增获得SU基因片段，将其克隆到pMD-19T载体；测序验证后，再亚克隆到PET-32a（+）上，在RosettagamiB（DE3）感受态细胞进行表达，采用SDS-PAGE 、Western-blot和ELISA对重组蛋白进行鉴定及抗原性分析。结果结果显示，表达的重组菌融合蛋白大小约64.38 kD，在IPTG终浓度0.4 mmol·L⁻¹、37 ℃诱导4 h表达效果最好。用纯化的ENTV-2进行SDS-PAGE，SU重组蛋白免疫小鼠制备的多克隆抗体作为一抗，进行 Western-blot，结果显示鼠抗SU蛋白多克隆抗体能与ENTV-2抗原进行特异性的反应。结论表达的SU蛋白有较好的抗原性，为制备ENTV-2特异性单克隆抗体及建立ENTV-2特异性血清学方法奠定了基础。
- 山羊地方性鼻内肿瘤病毒 /
- 表面蛋白 /
- 原核表达 /
- 多克隆抗体
Abstract: Objective In order to determine the function of SU protein of enzootic nasal tumor virus of goats (ENTV-2), Method RT-PCR method was used to amplify the SU gene fragment from ENTV-2 FJ and then the SU gene was cloned into pMD-19T Simple Vector; After sequencing, the cloning vector was subcloned into PET-32a (+), the recombinant plasmid was transformed into RosettagamiB (DE3) competent cells. SDS-PAGE, Western-blot and ELISA were used for identification and antigenicity analysis of recombinant proteins. Result The result showed that the expressed recombinant protein was about 64.38 kD, and the best expression condition was induced at 37 ℃ for 4 h at a final IPTG concentration of 0.4 mmol/L. The purified ENTV-2 virus was used for SDS-PAGE, and the mice anti-SU polyclonal antibody was used as primary antibody for Western-blot. The Western-blot analysis showed the mice anti-SU polyclonal antibody could react with ENTV-2 antigen specifically. It is proved that the expressed SU recombinant protein has better antigenicity. Conclusion The results proved that the expressed SU recombinant protein has better antigenicity, and provided a basis for the preparation of ENTV-2 specific monoclonal antibody and the establishment of ENTV-2 specific serological method.
- Enzootic nasal tumor virus of goats /
- surface protein /
- prokaryotic expression /
- polyclonal antibody

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图 1 SU基因RT-PCR扩增结果

注：M:DL2 000 DNA Marker 1:SU基因PCR扩增片段。

Figure 1. RT-PCR amplification result of SU gene

Note: M: DL2 000 DNA Marker ; 1:PCR amplification results of SU gene.

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图 2 SU-19T质粒双酶切结果

注：M:DL10 000 DNA Marker; 1:SU-19T质粒双酶切。

Figure 2. Double digestion result of SU-19T plasmid

Note: M: DL10 000 DNA Marker; 1: double digestion of SU-19T plasmid.

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图 3 PET32 a（+）质粒双酶切结果

注：M:DL10 000 DNA Marker ；1: PET32 a（+）质粒双酶切。

Figure 3. Double digestion result of PET32 a（+）

Note: M: DL10 000 DNA Marker; 1: double digestion of PET32 a（+）.

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图 4 不同IPTG诱导浓度对SU重组蛋白诱导表达的影响

注：M为蛋白Marker; 1为pET-32a(+)空载；2-7为菌液IPTG诱导终浓度分别为0.2、0.4、0.6、0.8、1.0、1.2 mmol·L^-1。

Figure 4. Effect of IPTG at different concentrations on the induced expression of SU recombinant protein

Note: M: Protein Marker; 1:Un-induced pET-32a(+) / RosettagamiB（DE3）; 2-7: pET-32a(+)-SU / RosettagamiB（DE3）induced with 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mmol·L^-1 IPTG, respectively.

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图 5 不同IPTG诱导时间对SU重组蛋白诱导表达的影响

注：M为蛋白Marker; 1为pET-32a(+)空载；2~6为菌液IPTG诱导时间分别为3、4、5、6h、过夜。

Figure 5. Effect of different IPTG induction time on the expression of SU recombinant protein

Note: M:Protein Marker; 1:Un-induced pET-32a(+) / RosettagamiB（DE3）; 2-6: pET-32a(+)-SU/RosettagamiB（DE3）induced for 3, 4, 5, 6 h and overnight, respectively.

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图 6 SU重组蛋白纯化结果

注：M为蛋白Marker; 1为未纯化蛋白；2为纯化蛋白。

Figure 6. The purification result of SU fusion protein

Note: M: Protein Marker; 1:unpurified protein; 2: purified protein.

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图 7 Western-blot 分析结果

注：M为蛋白Marker; （图A）1：一抗为正常对照小鼠血清（空白对照）；（图B）2：一抗为鼠抗纯化ENTV-2多抗（阳性对照）；（图C）3：一抗为鼠抗SU蛋白多抗。

Figure 7. The result of Western-blot analysis

Note: M:Protein Marker;（Fig A）1:the primary antibody is the serum of normal control mice （blank control）; （Fig B）2:the primary antibody is the mouse anti purified ENTV-2 polyclonal antibody （positive control）; （Fig C）3:the primary antibody is the mouse anti-SU protein polyclonal antibody.

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表 1 引物设计

Table 1. Primer design

引物名称 Primer name	引物序列 primer sequence	酶切位点 restriction enzyme cutting site
ENTV-2SU（F）	5′-CCGGAATTCTTTGTGTTTTTC CGCAGGAT-3′	ECORⅠ
ENTV-2SU（R）	5′-CCAAGCTTGCTCACACTTCT TTCCTTGC-3′	Hind Ⅲ

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表 2 ELISA试验结果

Table 2. ELISA result

抗体 antibody	包被抗原 coating antigen
抗体 antibody	ENTV-2	SU
SU（1∶4）	+	+
SU（1∶8）	−	+
SU（1∶16）	−	+
SU（1∶32）	−	+
ENTV-2（1∶4）	+	+
ENTV-2 （1:8）	+	+
ENTV-2 （1:16）	+	+
ENTV-2 （1:32）	+	+
注：“+”表示结果为阳性；“−”表示结果为阴性。 Note: “ +” means the result is positive; “−“ means the result is negative.

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