茶毛虫核型多角体病毒ph基因抗体的制备与利用

doi:10.13305/j.cnki.jts.2008.04.005

Abstract

Abstract: The 741 bp coding region of polyhedrin gene (ph) was amplified from Euproctis pseudoconspersa nucleopolyhedrovirus（EupsNPV）genome and its prokaryotic expression vector pET-28-a-Ep-ph was constructed. Then the prokaryotic expression plasmid was transformed into Escherichia.coli. BL21（DE3）for expression. The expressed fusion protein was separated by SDS-PAGE and retrieved from the gel. New Zealand white rabbit was immunized with the purified fusion protein and the antiserum against ph with a titre of 6.4×10⁴ was prepared. Western blotting indicated that the prepared polyclonal antibody was specific for polyhedrin. The EupsNPV was detected by indirect ELISA and its regression equation was obtained: y=0.4152x-0.8299 with correlation coeffient r=0.9897(P<0.01). Indirect ELISA detection showed that the antibody could be used for quantitative detection of the NPV in a commercial bio-pesticide and provided an accurate and effective method for detection.

Key words: Euproctis pseudoconspersa nucleopolyhedrovirus（EupsNPV）, polyhedrin gene, polyclonal antibody, indirect ELISA, detection

CLC Number:

S435.711

WANG Li-zhong, XIAO Qiang, ZHANG Chuan-xi. Preparation of the Antibody Against EupsNPV Polyhedrin and Its Utilization in Detection of the Viral Pesticide[J]. Journal of Tea Science, 2008, 28(4): 260-266.

References

[1] Blissard G, Black B, Crook N, et al.Family Baculoviridae. In: van Regenmortel M H V, Fauquet C M, Bishop D H L, et al. (Eds), Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses[C]. San Diego: Academic Press, 2000: 195~202.
[2] George F, Rohrmann. Polyhedrin Structure[J]. Journal of General Virology, 1986, 67: 1499~1513.
[3] George F, Rohrmann. Baculovirus structural proteins[J]. Journal of General Virology, 1992, 73: 749~761.
[4] 石正丽, 张立人, 陈棣华. 茶毛虫核型多角体病毒的血清学特性[J]. 中国病毒学, 1992, 7(3): 276~282.
[5] 陈海雷, 叶林柏, 刘年翠. 用微核试验测定几种茶树昆虫病毒对小白鼠的安全性[J]. 病毒学杂志, 1991, 6(2): 147~150.
[6] 冷杨, 殷坤山, 肖强. 茶毛虫病毒研究进展[J]. 茶叶, 2006, 32(2): 70~74.
[7] 厉晓腊, 金轶伟, 柴一秋, 等. 茶毛虫核型多角体病毒对茶毛虫的致病性研究[J]. 茶叶科学, 2006, 26(4): 265~269.
[8] 孙俶德, 吴光远, 林阿祥, 等. 茶毛虫核型多角体病毒制剂的防效[J]. 华东昆虫学报, 1996, 5(1):55~59.
[9] 殷坤山, 肖强. 茶毛虫病毒杀虫剂田间使用技术研究[J]. 中国茶叶, 2004, 26(3): 189~192.
[10] 陈棣华, 赵怀宇, 张立人. 三种昆虫核型多角体病毒DNA的限制性内切酶酶切分析[J]. 病毒学报, 1987, 3(1): 59~75.
[11] 陈绳亮, 罗保君, 常国辉, 等. 茶毛虫核型多角体病毒基因组酶切分析及质粒文库的构建[J]. 华中农业大学学报, 1999, 18(4): 303~306.
[12] 常国辉, 陈绳亮, 罗保君, 等. 茶毛虫核型多角体病毒(EupsNPV)多角体蛋白基因的定位及克隆[J]. 中国病毒学, 2001, 16(4): 390~392.
[13] 聂婷婷, 肖强, 殷坤山, 等. 茶毛虫NPV的P24、Rr1、Lef1基因及其分子进化分析[J]. 茶叶科学,2005, 25(4): 242~248.
[14] 张传溪, 孙建新, 王根, 等. EoNPV的限制性酶切图谱及polyhedrin基因和egt基因的定位[J]. 应用与环境生物学报, 1998, 4(1): 55~59.
[15] 张传溪. 昆虫杆状病毒ph、pk基因分析和人EPO基因在杆状病毒-昆虫系统中的表达[M]. 北京:高等教育出版社, 2001.
[16] Harlow E, Lane D.Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press[M]. Cold Spring Harbor, New York, USA, 1988.
[17] 于涟. 免疫学实验技术[M]. 浙江大学出版社, 1992.
[18] William H R, Langridge, Robert R, et al. Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)[J]. Journal of Invertebrate Pathology, 1981, 38(2): 242~250.
[19] R R DE Moraes, J E Maruniak, J E Funderburk. Methods for detection of Anticarsia gemmatalis Nucleopolyhedrovirus DNA in soil[J]. Applied and environmental microbiology, 1999, 65(6): 2307~2311.
[20] L S England, M L Vincent, J T Trevors, et al. Extraction and detection of baculoviral DNA from lake water, detritus and forest litter England LS[J]. Journal Applied Microbiology, 2001, 90: 630~636.
[21] Peter M Ebling,Stephen B Holmes.A refined method for the detection of baculovirus occlusion bodies in forest terrestrial and aquatic habitats[J]. Pest Management Science, 2002, 58 :1216~1222.
[22] 欧阳松应, 杨冬, 欧阳红生, 等. 生命的化学[M]. 2004, 24(1): 74~76.

Preparation of the Antibody Against EupsNPV Polyhedrin and Its Utilization in Detection of the Viral Pesticide

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