中蜂囊状幼虫病毒辽宁毒株的结构蛋白鉴定及杆状病毒表达载体的构建
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摘要
目的
本研究通过对中蜂囊状幼虫病毒辽宁株(CSBV-LN)进行提纯,SDS-PAGE检测和质谱分析,对CSBV-LN结构蛋白进行了鉴定。在此基础上,构建VP1结构蛋白的杆状病毒表达载体,并筛选出能高效表达VP1蛋白重组杆状病毒,该研究为进一步研制中蜂囊状幼虫病的血清诊断试剂盒奠定基础。
方法
采用差速离心法对CSBV-LN进行提纯,对纯化后的CSBV-LN进行SDS-PAGE,分离到4个蛋白,对分离到的4个蛋白进行质谱分析,鉴定出VP1、VP3和VP0三个蛋白。然后,对其中VP1蛋白的基因,进行克隆,测序正确后,克隆至杆状病毒表达载体的pFastBacI的BamHⅠ和HindⅢ位点,构建VP1基因的杆状病毒重组表达载体VP1-pFastBacI。将VP1-pFastBacI在DH10Bac里面进行转座,形成重组VP1-Bacmid,将杆粒转染至sf9昆虫细胞,重组得到含有VP1基因的杆状病毒,进行连续传代,用电镜观察、PCR和Western-blotting等检测方法,对重组病毒进行鉴定。
结果
1、对CSBV-LN进行了提纯,得到了CSBV-LN的病毒原液。
2、纯化后的CSBV-LN通过SDS-PAGE,分离到4个蛋白。对分离到的4个蛋白进行质谱分析,结果鉴定出VP1、VP3和VP0三个蛋白。
3、构建CSBV-LN结构蛋白VP1杆状病毒表达载体VP1-pFastBacI,并在大肠杆菌DH10Bac里进行了转座,得到重组载体VP1-Bacmid。
4、将构建出的重组载体VP1-Bacmid转染sf9昆虫细胞,收集病变细胞做Western-blotting检测,可检测出目的条带,大小约为31 KD。
结论
1、成功对CSBV-LN进行了提纯,得到了高纯度和高浓度的病毒原液。
2、通过对CSBV-LN进行SDS-PAGE以及蛋白的质谱分析,鉴定出VP1、VP3和VP0三个蛋白。
3、成功构建出CSBV-LN结构蛋白VP1杆状病毒表达载体VP1-pFastBacI。
4、成功构建了VP1杆状病毒重组载体VP1-Bacmid,并实现了VP1在重组杆状病毒的表达,筛选到一株能够表达VP1基因的重组杆状病毒。
Objective
Chinese sacbrood virus Liaoning strain (CSBV-LN) was purified and separated by SDS-PAGE, and the structural proteins were identified by mass spectrographic. In this base, the vector of VP1 gene was structured and the VP1 gene was expressed by the baculovirus expression system. This study is the basis for the further research of serodiagnosis kit of CSBV-LN.
Methods
CSBV-LN was purified from diseased insects. The four main structural of CSBV were separated by SDS-PAGE. Structural proteins VP1, VP3 and VP0 were identified by mass spectrographic. The VP1 protein was cloned and sequenced to construct baculovirus expression vector, then transfected into sf9 insect cell. The protein which was secreted by sf9 insect cell was detected by Western-blotting.
Results
1. CSBV-LN virus prefiltration was got by purification.
2. CSBV-LN was purified from diseased insects and was detected by SDS-PAGE from which structural proteins, VP1, VP3 and VP0, were identified after four proteins was analysed by mass spectrographic.
3. The baculovirus expression system: VP1-pFastBacI was structured and the transposition was occurred in DH10Bac, and the recombinant vector: VP1-Bacmid was got.
4. The recombination transposition vector was transfected into sf9 insect cell, and the diseased cells were collected to detect by Western-blotting. The objective band is about 31KD.
Conclusions
1. CSBV-LN was purified, and the virus prefiltration was high purity and high concentration.
2. CSBV-LN was purified from diseased insects and was detected by SDS-PAGE from which structural proteins, VP1, VP3 and VP0, were identified after four proteins was analysed by mass spectrographic.
3. The baculovirus expression system: VP1-pFastBacI was structured.
4. The baculovirus expression vector of VP1 was structured and was expressed in the recombinant baculovirus, and a strain of recombinant baculovirus was get.
本研究通过对中蜂囊状幼虫病毒辽宁株(CSBV-LN)进行提纯,SDS-PAGE检测和质谱分析,对CSBV-LN结构蛋白进行了鉴定。在此基础上,构建VP1结构蛋白的杆状病毒表达载体,并筛选出能高效表达VP1蛋白重组杆状病毒,该研究为进一步研制中蜂囊状幼虫病的血清诊断试剂盒奠定基础。
方法
采用差速离心法对CSBV-LN进行提纯,对纯化后的CSBV-LN进行SDS-PAGE,分离到4个蛋白,对分离到的4个蛋白进行质谱分析,鉴定出VP1、VP3和VP0三个蛋白。然后,对其中VP1蛋白的基因,进行克隆,测序正确后,克隆至杆状病毒表达载体的pFastBacI的BamHⅠ和HindⅢ位点,构建VP1基因的杆状病毒重组表达载体VP1-pFastBacI。将VP1-pFastBacI在DH10Bac里面进行转座,形成重组VP1-Bacmid,将杆粒转染至sf9昆虫细胞,重组得到含有VP1基因的杆状病毒,进行连续传代,用电镜观察、PCR和Western-blotting等检测方法,对重组病毒进行鉴定。
结果
1、对CSBV-LN进行了提纯,得到了CSBV-LN的病毒原液。
2、纯化后的CSBV-LN通过SDS-PAGE,分离到4个蛋白。对分离到的4个蛋白进行质谱分析,结果鉴定出VP1、VP3和VP0三个蛋白。
3、构建CSBV-LN结构蛋白VP1杆状病毒表达载体VP1-pFastBacI,并在大肠杆菌DH10Bac里进行了转座,得到重组载体VP1-Bacmid。
4、将构建出的重组载体VP1-Bacmid转染sf9昆虫细胞,收集病变细胞做Western-blotting检测,可检测出目的条带,大小约为31 KD。
结论
1、成功对CSBV-LN进行了提纯,得到了高纯度和高浓度的病毒原液。
2、通过对CSBV-LN进行SDS-PAGE以及蛋白的质谱分析,鉴定出VP1、VP3和VP0三个蛋白。
3、成功构建出CSBV-LN结构蛋白VP1杆状病毒表达载体VP1-pFastBacI。
4、成功构建了VP1杆状病毒重组载体VP1-Bacmid,并实现了VP1在重组杆状病毒的表达,筛选到一株能够表达VP1基因的重组杆状病毒。
Objective
Chinese sacbrood virus Liaoning strain (CSBV-LN) was purified and separated by SDS-PAGE, and the structural proteins were identified by mass spectrographic. In this base, the vector of VP1 gene was structured and the VP1 gene was expressed by the baculovirus expression system. This study is the basis for the further research of serodiagnosis kit of CSBV-LN.
Methods
CSBV-LN was purified from diseased insects. The four main structural of CSBV were separated by SDS-PAGE. Structural proteins VP1, VP3 and VP0 were identified by mass spectrographic. The VP1 protein was cloned and sequenced to construct baculovirus expression vector, then transfected into sf9 insect cell. The protein which was secreted by sf9 insect cell was detected by Western-blotting.
Results
1. CSBV-LN virus prefiltration was got by purification.
2. CSBV-LN was purified from diseased insects and was detected by SDS-PAGE from which structural proteins, VP1, VP3 and VP0, were identified after four proteins was analysed by mass spectrographic.
3. The baculovirus expression system: VP1-pFastBacI was structured and the transposition was occurred in DH10Bac, and the recombinant vector: VP1-Bacmid was got.
4. The recombination transposition vector was transfected into sf9 insect cell, and the diseased cells were collected to detect by Western-blotting. The objective band is about 31KD.
Conclusions
1. CSBV-LN was purified, and the virus prefiltration was high purity and high concentration.
2. CSBV-LN was purified from diseased insects and was detected by SDS-PAGE from which structural proteins, VP1, VP3 and VP0, were identified after four proteins was analysed by mass spectrographic.
3. The baculovirus expression system: VP1-pFastBacI was structured.
4. The baculovirus expression vector of VP1 was structured and was expressed in the recombinant baculovirus, and a strain of recombinant baculovirus was get.
引文
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3 Rossmanm MG, Arnold E, Erickson J, et al. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature, 1985, 317: 145-153.
4 Scraba DG. Functional aspects of the capsid structure of Mengo virus. J Struct Biol, 1990, 104: 52-62.
5 Acharya R, Fry E, Stuart D, et al. The three-dimensional structure of foot-and-mouth disease virus at 2.9A resolution. Nature, 1989, 337: 709-716.
6 Tate J, Liljas L, Scotti P. The crystal structure of cricket paralysis virus: the first view of a new family. Nature Struct Biol, 1999, 6: 765-774.
7冯建勋,卢炘英,张勤奋,等.中蜂囊状幼虫病病毒的纯化、结晶和结构研究.电子显微学报, 1998, 17(4): 387-388.
8张景强,冯建勋,梁玉尧,等.中蜂囊状幼虫病病毒的三维结构.中国科学, 2001, 31(4): 367-371.
9 Allen MF, Ball BV. The incidence and world distribution of honey bee viruses. Bee World, 1996, 77: 141-162.
10 Dall DJ. Inapparent infection of honey bee pupae by Kashmir and sacbrood bee viruses in Australia. Ann Appl Biol, 1985, 106(3): 461-468.
11 Grabensteiner E, Ritter W, Carter MJ, et al. Sacbrood virus of the honeybee (Apis mellifera): rapid identification and phylogenetic analysis using reverse transcription-PCR. Clin Diagn Lab Immunol, 2001, 8(1): 93-104.
12 Tentcheva D, Gauthier L, Zappulla N, et al. Prevalence and seasonal variation of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France. Appl Environ Microbiol, 2004, 70(12): 7185-7191.
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15 Zhang QF, Yang YF, Liang YY, et al. Study on structure of the Chinese Sacbrood Virus’nucleic acid. Journal of Chinese Electron Microscopy Society, 2002, 21(3): 331-334.
16董秉义,袁跃东,张弓绍,等.中蜂囊状幼虫病病毒的电镜观察.中国蜂业, 1984, 3: 8-9.
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18张景强,冯建勋,梁玉尧,等.中蜂囊状幼虫病病毒的三维结构.中国科学, 2001, 31(4): 367-371.
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21 Hinton TM, Ross-Smith N, Warner S, et al. Conservation of L and 3C proteinase activities across distantly related aphthoviruses. Journal of General Virology, 2002, (12): 3111-3121.
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3 Grabensteiner E, Ritter W, Carter MJ, et al. Sacbrood virus of the honeybee(Apis mellifera): rapid identification and phylogenetic analysis using reverse transcription-PCR. Clin Diagn Lab Immunol, 2001, 8(1): 93-104.
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37 Lanzi G, de Miranda JR, Boniotti MB, et al. Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L.). Jounal of Virology, 2006, 80(10): 4988-5009.
38 Hinton TM, Ross-Smith N, Warner S, et al. Conservation of L and 3C proteinase activities across distantly related aphthoviruses. Journal of General Virology, 2002, 83: 3111-3121.
39 Ma MX, Li M, Cheng J, et al. Molecular and Biological Characterization of Chinese Sacbrood Virus LN Isolate. Comparative and Functional Genomics, 2011, 2011: 409386.
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2 Hogle JM, Chou M, Fijman DJ. Three-dimensional structure of poliovirus at 2.9A resolution. Science, 1985, 229: 1358-1365.
3 Rossmanm MG, Arnold E, Erickson J, et al. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature, 1985, 317: 145-153.
4 Scraba DG. Functional aspects of the capsid structure of Mengo virus. J Struct Biol, 1990, 104: 52-62.
5 Acharya R, Fry E, Stuart D, et al. The three-dimensional structure of foot-and-mouth disease virus at 2.9A resolution. Nature, 1989, 337: 709-716.
6 Tate J, Liljas L, Scotti P. The crystal structure of cricket paralysis virus: the first view of a new family. Nature Struct Biol, 1999, 6: 765-774.
7冯建勋,卢炘英,张勤奋,等.中蜂囊状幼虫病病毒的纯化、结晶和结构研究.电子显微学报, 1998, 17(4): 387-388.
8张景强,冯建勋,梁玉尧,等.中蜂囊状幼虫病病毒的三维结构.中国科学, 2001, 31(4): 367-371.
9 Allen MF, Ball BV. The incidence and world distribution of honey bee viruses. Bee World, 1996, 77: 141-162.
10 Dall DJ. Inapparent infection of honey bee pupae by Kashmir and sacbrood bee viruses in Australia. Ann Appl Biol, 1985, 106(3): 461-468.
11 Grabensteiner E, Ritter W, Carter MJ, et al. Sacbrood virus of the honeybee (Apis mellifera): rapid identification and phylogenetic analysis using reverse transcription-PCR. Clin Diagn Lab Immunol, 2001, 8(1): 93-104.
12 Tentcheva D, Gauthier L, Zappulla N, et al. Prevalence and seasonal variation of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France. Appl Environ Microbiol, 2004, 70(12): 7185-7191.
13张忠信,王瑶.病毒结构研究?病毒分类学?北京:高等教育出版社, 2006?13.
14 Ghosh RC, Ball BV, Willcocks MM, et al. The nucleotide sequence of sacbrood virus ofthe honey bee: an insect picorna-like virus. J Gen Virol, 1999, 80: 1541-1549.
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