油桐尺蠖核型多角体病毒（BusuNPV）分离株比较及微量检测方法的研究
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摘要
本论文对油桐尺蠖核型多角体病毒(Buzura suppressatia nucleopolydrovirus,BusuNPV)的不同毒株分别通过生物测定,了解不同毒株的毒力大小;制作超薄切片,在扫描电镜下观察不同毒株的外部形态特征,在透射电镜下观察其病毒粒子的分布状况;病毒基因组酶切分析病毒核酸的变化。根据油桐尺蠖核型多角体病毒(BusuNPV)的多角体囊膜蛋白基因(pep)、lef-2基因设计特异性引物,成功建立了BusuNPV的PCR检测技术体系。研究内容与结果如下:
(1)对BusuNPV各分离株进行毒力测定,未见明显差别。LF、SF、WF的LC50分别为4.39×104、6.65×104、1.02×105PIB/ml;LT50分别为5.59、5.69、5.17d。观察扫描电镜、透射电镜照片,发现各分离株均为单粒包埋核型多角体病毒,多角体直径为0.85-1.50um,病毒粒子大小为0.195-0.246 um×0.054-0.072 um,各毒株间未见显著差别。
(2)选择5种常见内切酶对BusuNPV全基因组序列进行酶解,除EcoRⅠ外其它内切酶酶解条带均较少,分子量大,不易进行分离株比较。其中EcoRⅠ消化的DNA片段约为12条,分子量小于15kb,分离株之间未见差别。双酶酶解BusuNPV全基因组序列时,DNA充分被消化,条带较多,适宜分离株之间比较。但是观察双酶切图谱,各分离株的酶切条带数目、大小不存在差别。
(3)通过基因比对,分别确定了BusuNPV的两个特异性片段pep基因和lef-2基因,并根据两段基因设计出两对特异性引物BPF、BLA,建立以PCR技术为基础的快速检测BusuNPV的方法。
(4)建立的BusuNPV的PCR检测技术,不仅能短时间内成功的从卵和蛹内检测到BusuNPV,而且其灵敏度可达到1 fg·ml -1DNA。经过模板多样性研究,用多角体悬液也可直接作为模板进行PCR试验检测BusuNPV。
(5)本实验所建立的BusuNPV PCR快速检测技术,检测的特异性强、灵敏度高、操作简单易行,且应用效果非常好,可以用来研究病毒在虫体内的增殖规律及检测林间油桐尺蠖的带毒情况。
The thesis made the test by biological in order to know the virulence of different strains of Buzura suppressatia nucleopolydrovirus, to survey external characteristics of different strains with scanning electron microscopy and distribution of virus particles with transmission electron microscope by production of thin sections; virus genome restriction enzyme analysis of viral nucleic acid changes. To design specific primers according to the polyhedrin envelope protein gene (pep) and lef-2 gene of BusuNPV, the PCR detection technology of BusuNPV successfully established. The study and results are as follows:
(1)There is no significant difference virulence of all BusuNPV isolates by measured. The LC50 of LF, SF, and WF were 4.39×104, 6.65×104 and 1.02×105PIB/ml; the LT50 were 5.59, 5.69, and 5.17 d. It is showed that all isolates are single-particle-embedded nuclear polyhedrosis virus by observing photographs of scanning electron microscopy and transmission electron microscope, polyhedron diameter is 0.85-1.50um, the virus particle size is 0.195-0.246 um×0.054-0.072 um.
(2)Digesting the whole genome sequence of BusuNPV by selecting 5 kinds of enzymes. In addition to EcoRⅠ,endonuclease digestion of foreign bands are small, molecular weight, which is difficult to comprising the isolate. Digestion of DNA fragments by EcoRⅠwas about 12, the molecular weight was less than 15kb, there is no differences in isolates. Digesting the BusuNPV by double enzyme genome sequence, DNA was fully digested bands are more suitable for comparison in isolates. However, restriction enzyme digestion patterns observed, the isolated enzyme band number and size have no different.
(3)Identifying two specific gene fragments pep and lef-2 of BusuNPV by comparing gene, and designing two pairs of specific primers BPF and BLA. Establishing a rapid detection BusuNPV method based PCR technology.
(4)PCR detection technology of BusuNPV is not only detecting BusuNPV from the eggs and the pupae successfully within a short time ,but also its sensitivity was 1 fg/mlDNA. After the template diversity, polyhedral suspension can also be used directly as template for PCR technology to detect BusuNPV.
(5)Established PCR technology in experiment was rapid, specific, sensitive, simple and the application results was very good. It can be used to study the proliferation disciplinarian of the virus in the insect body and the detecting situation of virus in the insect body in forest.
(1)对BusuNPV各分离株进行毒力测定,未见明显差别。LF、SF、WF的LC50分别为4.39×104、6.65×104、1.02×105PIB/ml;LT50分别为5.59、5.69、5.17d。观察扫描电镜、透射电镜照片,发现各分离株均为单粒包埋核型多角体病毒,多角体直径为0.85-1.50um,病毒粒子大小为0.195-0.246 um×0.054-0.072 um,各毒株间未见显著差别。
(2)选择5种常见内切酶对BusuNPV全基因组序列进行酶解,除EcoRⅠ外其它内切酶酶解条带均较少,分子量大,不易进行分离株比较。其中EcoRⅠ消化的DNA片段约为12条,分子量小于15kb,分离株之间未见差别。双酶酶解BusuNPV全基因组序列时,DNA充分被消化,条带较多,适宜分离株之间比较。但是观察双酶切图谱,各分离株的酶切条带数目、大小不存在差别。
(3)通过基因比对,分别确定了BusuNPV的两个特异性片段pep基因和lef-2基因,并根据两段基因设计出两对特异性引物BPF、BLA,建立以PCR技术为基础的快速检测BusuNPV的方法。
(4)建立的BusuNPV的PCR检测技术,不仅能短时间内成功的从卵和蛹内检测到BusuNPV,而且其灵敏度可达到1 fg·ml -1DNA。经过模板多样性研究,用多角体悬液也可直接作为模板进行PCR试验检测BusuNPV。
(5)本实验所建立的BusuNPV PCR快速检测技术,检测的特异性强、灵敏度高、操作简单易行,且应用效果非常好,可以用来研究病毒在虫体内的增殖规律及检测林间油桐尺蠖的带毒情况。
The thesis made the test by biological in order to know the virulence of different strains of Buzura suppressatia nucleopolydrovirus, to survey external characteristics of different strains with scanning electron microscopy and distribution of virus particles with transmission electron microscope by production of thin sections; virus genome restriction enzyme analysis of viral nucleic acid changes. To design specific primers according to the polyhedrin envelope protein gene (pep) and lef-2 gene of BusuNPV, the PCR detection technology of BusuNPV successfully established. The study and results are as follows:
(1)There is no significant difference virulence of all BusuNPV isolates by measured. The LC50 of LF, SF, and WF were 4.39×104, 6.65×104 and 1.02×105PIB/ml; the LT50 were 5.59, 5.69, and 5.17 d. It is showed that all isolates are single-particle-embedded nuclear polyhedrosis virus by observing photographs of scanning electron microscopy and transmission electron microscope, polyhedron diameter is 0.85-1.50um, the virus particle size is 0.195-0.246 um×0.054-0.072 um.
(2)Digesting the whole genome sequence of BusuNPV by selecting 5 kinds of enzymes. In addition to EcoRⅠ,endonuclease digestion of foreign bands are small, molecular weight, which is difficult to comprising the isolate. Digestion of DNA fragments by EcoRⅠwas about 12, the molecular weight was less than 15kb, there is no differences in isolates. Digesting the BusuNPV by double enzyme genome sequence, DNA was fully digested bands are more suitable for comparison in isolates. However, restriction enzyme digestion patterns observed, the isolated enzyme band number and size have no different.
(3)Identifying two specific gene fragments pep and lef-2 of BusuNPV by comparing gene, and designing two pairs of specific primers BPF and BLA. Establishing a rapid detection BusuNPV method based PCR technology.
(4)PCR detection technology of BusuNPV is not only detecting BusuNPV from the eggs and the pupae successfully within a short time ,but also its sensitivity was 1 fg/mlDNA. After the template diversity, polyhedral suspension can also be used directly as template for PCR technology to detect BusuNPV.
(5)Established PCR technology in experiment was rapid, specific, sensitive, simple and the application results was very good. It can be used to study the proliferation disciplinarian of the virus in the insect body and the detecting situation of virus in the insect body in forest.
引文
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