家蚕浓核病毒BmDNV-1荧光假病毒粒子的制备及人博卡病毒VP2的纯化
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摘要
导致人类疾病的病原体在不断进化,人类生产方式和生活环境的巨大演变也加速着这一进化过程。新病毒以及新型疾病的出现给人类生命健康和传统疾病治疗手段带来了前所未有的挑战,如2003年在亚洲急剧流行的严重急性呼吸道综合症(SARS)、2009年席卷全球的甲型流感病毒(H1N1、H5N1)和2005年新发现的细小病毒科人博卡病毒(HBoV)等。在众多威胁人类健康的疾病之中,恶性肿瘤(又称癌症)依旧占据着主要地位,并且尚无切实可行的治疗方法。
寻找可行的癌症治疗手段和方法,已经引起了医学界和科学界的密切关注,人们不断探索着一些新的治疗方法。相对于传统癌症治疗手段(化疗、放疗)所带来的严重副反应特性,近年来出现的生物纳米技术在癌症治疗方面展现出了巨大潜力和优势。纳米生物技术是运用一些纳米颗粒或类似于纳米颗粒的生物材料(如病毒样颗粒)作为载体平台,来实现对病变等非正常细胞的特异性进攻,以达到治疗疾病的目的。目前,有关病毒样颗粒在细胞内或生物机体内的转运途径与机制、病毒样颗粒体外去组装与重组装、生物效应分子的偶联与包被等特性尚不够清楚。
本研究基于已有有关细小病毒的研究,体外构建家蚕浓核病毒伊那株(BmDNV-1)结构蛋白基因vp4与增强型绿色荧光蛋白egfp基因融合表达载体,利用重组杆状病毒真核表达系统,制备荧光病毒样颗粒(fluorescent virus-like particle, fVLP)。得到的荧光病毒样颗粒因携带有绿色荧光蛋白标记,可以方便地用于其入胞、转运、定位以及体外自组装等生物学特性研究,为有关癌症治疗药物的研发提供参考。另外,以人博卡病毒(Human Bocavirus, HBoV-1)结构蛋白VP2为研究对象,构建VP2原核表达载体,亲和纯化VP2蛋白,用于多克隆抗体制备,用于人博卡病毒的快速检测和病毒蛋白亚细胞定位研究。
完成的工作包括以下几个方面：
1.制备家蚕浓核病毒结构蛋白VP4与EGFP融合表达的荧光病毒样颗粒
1)PCR分别体外扩增家蚕浓核病毒结构蛋白基因vp4和绿色荧光蛋白egfp基因,通过酶切、连接和转化等步骤筛选重组克隆。将重组质粒转化大肠杆菌DH10Bac,在协助质粒的帮助下,构建含结构基因vp4和egfp基因融合表达的重组Bacmid。
2)重组Bacmid转染Sf9昆虫细胞表达产生重组杆状病毒,获得重组杆状病毒病毒液。以最适感染复数的病毒液感染hi5细胞以表达荧光病毒样颗粒EGFP-VP4fVLP,然后利用蔗糖垫层及CsCl梯度离心获得较纯净的fVLP,电镜观察。
2.表达并纯化HBoV-1结构蛋白VP2
1)PCR扩增HBoV结构蛋白基因vp2,并将其分别连接到pET-28a(+)和pMAL-c2x原核表达载体上,获得原核表达重组质粒pET-28a(+)-VP2和pMAL-c2x-VP2,将重组质粒分别转入相应表达宿主菌,检测目的蛋白VP2表达情况。
2)将不同分子伴侣导入重组表达菌,检测目的蛋白的可溶性及表达情况,利用亲和树脂纯化VP2蛋白,纯化获得的VP2蛋白应用于多克隆抗体的制备。
Human pathogens which lead to diseases are always evolutionary. Environmental changements and living condition variations have speeded up this process. Emergent, re-emergent viruses and novel diseases caused by those viruses are great challenges to public health as well as classic disease treatments which humans have never suffered before. For instance, the outbreak of Serve Acute Respiratory Syndrome virus (SARS) in Asia in2003, the pandemic influenza virus A (H1N1, H5N1) in2009and the newly discovered Human Boca Virus (HBoV) within the family parvoviridae in2005are typical examples. Malignant tumor which called cancer also is one of the diseases which threat human health the most. Until now, there is no efficient treatment.
Looking for the valuable tools and methods to treat cancer is one of the hottest projects in scientific and medical fields. Scientists all the world are trying their best to find different ways to treat or prevent these diseases. The classical treatments of cancer, such as chemotherapy and radiotherapy, they are often accompanied with serious side effects. The newly emerged nano-biotechnology seems to have many potentials and advantages in cancer treatment. Nano-biotechnology utilizes nano materials or biological materials similar to nano particles as carrier, such as virus-like particle, to attack the abnormal cells specifically in order to cure diseases. However, the knowledge regarding to the pathway and mechanism of virus-like particle movement in cells or organisms, virus-like particle dis-assembling and re-assembling in vitro as well as the conjunction and embedding of biological effectors into the virus-like particle are not yet elucidated.
This project based on the knowledge and characteristics of parvoviruses. We constructed the fusion protein expression vector of BmDNV-1Ina isolate vp4gene with the coding sequence of enhanced green fluorescent protein (EGFP) in vitro, and then produced fluorescent virus-like particle (fVLP) using Bac-to-Bac recombinant baculovirus eukaryotic expression system. These fVLPs attached with EGFP fluorescent marker can be easily used to investigate its cell penetration, movement, location, together with the dis-assembling and re-assembling properties. These results can provide useful information about anti-cancer drug designs and developments. Besides, the Human bocavirus (HBoV) vp2gene is also a good candidate for construction of VPLs, therefore, we constructed the prokaryotic expression vector and purified VP2protein to prepare polyclonal antibody for fast detection of HBoV in cells which provides us a useful tool to investigate viral proteins in cellular distribution.
The main results of this project are summarized as below:
1. Fluorescent virus-like particle of BmDNV-1VP4and EGFP fusion protein production
1) The gene vp4of BmDNV-1and egfp gene were amplified respectively, followed by restriction enzyme digestion, ligation, transformation and screening to obtain the positive recombinants. Recombinant plasmid was transformed into E. coli DH10Bac competent cell. With the help of helper plasmid, the expression cassette was transferred and recombinant bacmid was obtained.
2) Recombinant bacmid was used to transfect sf9insect cell line to obtain recombinant baculovirus. The recombinant baculovirus was then applied to infect hi5insect cell line with the optimal ratio of baculovirus stock solution to insect cell in order to acquire the EGFP-VP4fVLP. Then sucrose gradient and CsCl gradient centrifugation were used to purify the fVLP, followed with TEM observation.
2. Expression and purification of HBoV VP2
1) HBoV structural gene vp2was amplified and inserted into prokaryotic expression vector pET-28a (+) and pMAL-c2x, the recombinant expression plasmids pET-28a (+)-VP2and pMAL-c2x-VP2were acquired and then transformed into host bacteria for protein expression and the expression levels were assayed.
2) Different molecular chaperons were introduced into the host bacteria; the solubility and expression level of target protein were analyzed. The target protein was purified by affinity chromatography, which was used for polyclonal antibody production.
寻找可行的癌症治疗手段和方法,已经引起了医学界和科学界的密切关注,人们不断探索着一些新的治疗方法。相对于传统癌症治疗手段(化疗、放疗)所带来的严重副反应特性,近年来出现的生物纳米技术在癌症治疗方面展现出了巨大潜力和优势。纳米生物技术是运用一些纳米颗粒或类似于纳米颗粒的生物材料(如病毒样颗粒)作为载体平台,来实现对病变等非正常细胞的特异性进攻,以达到治疗疾病的目的。目前,有关病毒样颗粒在细胞内或生物机体内的转运途径与机制、病毒样颗粒体外去组装与重组装、生物效应分子的偶联与包被等特性尚不够清楚。
本研究基于已有有关细小病毒的研究,体外构建家蚕浓核病毒伊那株(BmDNV-1)结构蛋白基因vp4与增强型绿色荧光蛋白egfp基因融合表达载体,利用重组杆状病毒真核表达系统,制备荧光病毒样颗粒(fluorescent virus-like particle, fVLP)。得到的荧光病毒样颗粒因携带有绿色荧光蛋白标记,可以方便地用于其入胞、转运、定位以及体外自组装等生物学特性研究,为有关癌症治疗药物的研发提供参考。另外,以人博卡病毒(Human Bocavirus, HBoV-1)结构蛋白VP2为研究对象,构建VP2原核表达载体,亲和纯化VP2蛋白,用于多克隆抗体制备,用于人博卡病毒的快速检测和病毒蛋白亚细胞定位研究。
完成的工作包括以下几个方面：
1.制备家蚕浓核病毒结构蛋白VP4与EGFP融合表达的荧光病毒样颗粒
1)PCR分别体外扩增家蚕浓核病毒结构蛋白基因vp4和绿色荧光蛋白egfp基因,通过酶切、连接和转化等步骤筛选重组克隆。将重组质粒转化大肠杆菌DH10Bac,在协助质粒的帮助下,构建含结构基因vp4和egfp基因融合表达的重组Bacmid。
2)重组Bacmid转染Sf9昆虫细胞表达产生重组杆状病毒,获得重组杆状病毒病毒液。以最适感染复数的病毒液感染hi5细胞以表达荧光病毒样颗粒EGFP-VP4fVLP,然后利用蔗糖垫层及CsCl梯度离心获得较纯净的fVLP,电镜观察。
2.表达并纯化HBoV-1结构蛋白VP2
1)PCR扩增HBoV结构蛋白基因vp2,并将其分别连接到pET-28a(+)和pMAL-c2x原核表达载体上,获得原核表达重组质粒pET-28a(+)-VP2和pMAL-c2x-VP2,将重组质粒分别转入相应表达宿主菌,检测目的蛋白VP2表达情况。
2)将不同分子伴侣导入重组表达菌,检测目的蛋白的可溶性及表达情况,利用亲和树脂纯化VP2蛋白,纯化获得的VP2蛋白应用于多克隆抗体的制备。
Human pathogens which lead to diseases are always evolutionary. Environmental changements and living condition variations have speeded up this process. Emergent, re-emergent viruses and novel diseases caused by those viruses are great challenges to public health as well as classic disease treatments which humans have never suffered before. For instance, the outbreak of Serve Acute Respiratory Syndrome virus (SARS) in Asia in2003, the pandemic influenza virus A (H1N1, H5N1) in2009and the newly discovered Human Boca Virus (HBoV) within the family parvoviridae in2005are typical examples. Malignant tumor which called cancer also is one of the diseases which threat human health the most. Until now, there is no efficient treatment.
Looking for the valuable tools and methods to treat cancer is one of the hottest projects in scientific and medical fields. Scientists all the world are trying their best to find different ways to treat or prevent these diseases. The classical treatments of cancer, such as chemotherapy and radiotherapy, they are often accompanied with serious side effects. The newly emerged nano-biotechnology seems to have many potentials and advantages in cancer treatment. Nano-biotechnology utilizes nano materials or biological materials similar to nano particles as carrier, such as virus-like particle, to attack the abnormal cells specifically in order to cure diseases. However, the knowledge regarding to the pathway and mechanism of virus-like particle movement in cells or organisms, virus-like particle dis-assembling and re-assembling in vitro as well as the conjunction and embedding of biological effectors into the virus-like particle are not yet elucidated.
This project based on the knowledge and characteristics of parvoviruses. We constructed the fusion protein expression vector of BmDNV-1Ina isolate vp4gene with the coding sequence of enhanced green fluorescent protein (EGFP) in vitro, and then produced fluorescent virus-like particle (fVLP) using Bac-to-Bac recombinant baculovirus eukaryotic expression system. These fVLPs attached with EGFP fluorescent marker can be easily used to investigate its cell penetration, movement, location, together with the dis-assembling and re-assembling properties. These results can provide useful information about anti-cancer drug designs and developments. Besides, the Human bocavirus (HBoV) vp2gene is also a good candidate for construction of VPLs, therefore, we constructed the prokaryotic expression vector and purified VP2protein to prepare polyclonal antibody for fast detection of HBoV in cells which provides us a useful tool to investigate viral proteins in cellular distribution.
The main results of this project are summarized as below:
1. Fluorescent virus-like particle of BmDNV-1VP4and EGFP fusion protein production
1) The gene vp4of BmDNV-1and egfp gene were amplified respectively, followed by restriction enzyme digestion, ligation, transformation and screening to obtain the positive recombinants. Recombinant plasmid was transformed into E. coli DH10Bac competent cell. With the help of helper plasmid, the expression cassette was transferred and recombinant bacmid was obtained.
2) Recombinant bacmid was used to transfect sf9insect cell line to obtain recombinant baculovirus. The recombinant baculovirus was then applied to infect hi5insect cell line with the optimal ratio of baculovirus stock solution to insect cell in order to acquire the EGFP-VP4fVLP. Then sucrose gradient and CsCl gradient centrifugation were used to purify the fVLP, followed with TEM observation.
2. Expression and purification of HBoV VP2
1) HBoV structural gene vp2was amplified and inserted into prokaryotic expression vector pET-28a (+) and pMAL-c2x, the recombinant expression plasmids pET-28a (+)-VP2and pMAL-c2x-VP2were acquired and then transformed into host bacteria for protein expression and the expression levels were assayed.
2) Different molecular chaperons were introduced into the host bacteria; the solubility and expression level of target protein were analyzed. The target protein was purified by affinity chromatography, which was used for polyclonal antibody production.
引文
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[2]Usategui-Gomez M, Toolan HW, Ledinko N, et al. Single-stranded DNA from the parvovirus, H-1. Virology,1969,39(3):617-621.
[3]Bourguignon GJ, Tattersall PJ, Ward DC, et al. DNA of minute virus of mice: self-priming, nonpermuted, single-stranded genome with a 5'-terminal hairpin duplex. Journal of Virology,1976,20(1):290-306.
[4]Faust EA, Rankin CD. In vitro conversion of MVM parvovirus single-stranded DNA to the replicative form by DNA polymerase a from Ehrlich ascites tumour cells. Nucleic Acids Research,1982,10(14):4181-4201.
[5]Virus Taxonomy:Ninth Report of the International Committee on Taxonomy of Viruses. San Diego, USA, Elsevier Academic Press,2011.
[6]Yasha Li, YanMing Dong, Jun Jiang, et al. High prevelance of human parvovirus infection in patients with malignant tumors. Oncology Letters,2012,3(3):635-640.
[7]Csagola A, Lorincz M, Cadar D, et al. Detection, prevalence and analysis of emerging porcine parvovirus infections. Arch Virol,2012, published ahead of print.
[8]Li Y, Zadori Z, Bando H, et al. Genome organization of the densovirus from Bombyx mori (BmDNV-1) and enzyme activity of its capsid. Journal of General Virology, 2001,82(11):2821-2825.
[9]Bando H, Choi H, Ito Y, Kawase S. Terminal structure of a densovirus implies a hairpin transfer replication which is similar to the model for AAV. Virology,1990, 179(1):57-63.
[10]Bando H, Choi H, Ito Y, et al. Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm:the DNA molecules share a common terminal sequence. Archives of Virology,1992,124(1-2):187-193.
[11]Wang YJ, Yao Q, Chen KP, et al. Characterization of the genome structure of Bombyx mori densovirus (China isolate). Virus Genes,2007,35(1):103-108.
[12]Kidokoro K, Ito K, Ogoyi DO, et al. Non-susceptibility genes to Bombyx densovirus type 1, Nid-1 and nsd-1, affect distinct steps of the viral infection pathway. Journal of Invertebrate Pathology,2010,103(1):79-81.
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