家蚕新型高效杆状病毒表达系统的开发和应用研究
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摘要
昆虫杆状病毒表达载体系统(Baculovirus Expression Vector System,简称BEVS)是利用携带有外源目的基因的重组昆虫杆状病毒作载体在昆虫体内或昆虫培养细胞进行表达生产的一个重组蛋白生产系统。由于该系统所需要的周期远比动物或植物系统短,可以利用昆虫个体或其培养细胞进行大规模的表达生产,生产的重组蛋白产量高,蛋白翻译后加工比细菌、酵母生产系统完善,而且由于昆虫杆状病毒具有限制性的宿主范围,只对特定种属的昆虫及其细胞进行感染,对人畜等脊椎动物没有感染能力,因此具有比在哺乳动物及其培养细胞生产系统更为安全等优点而成为目前最有效的真核表达系统之一。
目前昆虫杆状病毒表达系统因使用的病毒载体不同,主要可以分为两类,一类以苜蓿银纹夜蛾多核衣壳核型多角体病毒(Autographa californica multinucleocapsid nucleopolyhedrovirus,简写为AcMNPV)作为表达载体；另一类以家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,简写为BmNPV)作为表达载体。AcMNPV表达载体系统已经发展比较成熟,在欧美国家应用较多。与之不同,家蚕BmNPV由于可以用家蚕幼虫或蛹作为载体进行大规模的基因表达,蛋白质翻译后加工修饰完善,生产成本较低。特别在我国,家蚕是一种非常重要的特色经济资源昆虫,饲养技术非常成熟,在将来的基因工程产业化方面已显示出广阔的应用前景。
然而,家蚕BmNPV杆状病毒表达系统在技术上还存在许多欠缺,特别在进行大规模产业化开发方面还有一些技术瓶颈问题迫切需要解决,主要有以下几个方面：(1)重组病毒的构建过程繁琐,花费时间长。传统的重组BmNPV的构建和筛选方法还依赖于培养细胞内的基因同源重组和空斑分析,需要非常熟练的技巧,而且花费时间较长,通常需要2-3个月：(2)重组蛋白产物在后期常被降解,生产效率降低。杆状病毒表达系统是一个裂解性的系统,杆状病毒本身具有多个蛋白酶基因,在表达目的基因产物的同时,这些蛋白酶也得到了表达。特别是在感染晚期,重组蛋白易受蛋白酶降解,而感染的家蚕体内的蛋白质降解问题尤其严重；(3)目前感染接种采用的经皮接种技术,效率低下。为了达到较高的表达水平,目前构建重组病毒通常将目的基因替换多角体基因,从而形成不产生多角体的芽生型“裸露”重组病毒。重组病毒经口感染效率很低,因此常采用皮下注射感染的方法,不仅工效低,而且容易产生污染。
针对上述问题,本研究通过现代分子生物学技术和手段,借鉴国外经验,对家蚕杆状病毒表达系统进行了有力改造,使之成为一个更加高效的、能够适合产业化生产的表达系统。主要研究成果如下：[1]构建了适用于家蚕的BmNPV Bac-to-Bac/Bacmid快速高效表达系统。借鉴国外业已开发成功的AcMNPVBac-to-Bac快速基因表达系统工作原理,对家蚕BmNPV基因组进行了改造。通过同源重组的方法,将含有单拷贝数细菌F复制子、插入有细菌转座子整合靶位点、编码LacZa肽的部分DNA片段和抗性选择标记基因的基因片段重组入家蚕BmNPV基因组,替换多角体蛋白基因,获得了家蚕BmNPV病毒穿梭载体BmBacmid;并利用供体质粒上的表达盒和细菌转座子,和细菌转座子的基因定位转移作用,在细菌体内实现外源目的基因向家蚕BmNPV基因组上的转移整合,快速完成重组BmNPV病毒的构建。同时,对配套供体质粒进行了改造,构建带有分泌信号的供体质粒pBacGP67。[2]构建了能够表达多角体的重组BmNPV杆状病毒表达系统。将polh基因插入到p10位点,使用p10启动子控制多角体蛋白的表达。将筛选得到的p10被polh替换的Bmbacmid (polh+) DNA和helper质粒先后转化入DH10β,做成感受态细胞,得到同时含有Bmbacmid (polh+)和helper的DH10β,命名为DH10BmBac(polh+),结合上面构建的分泌型供体质粒,即构建成BmNPV Bac-to-Bac(polh+)快速基因表达系统。[3]删除了BmNPV杆状病毒自身的一种主要的蛋白酶基因,有效提高了表达效率。对杆状病毒基因组中引起蛋白降解的主要基因,半胱氨酸蛋白酶基因(CP),进行了基因敲除。同时,为了充分发挥本实验室构建的HyNPV病毒的优势,我们将Bac-to-Bac系统中的关键的8.6 kb片段按照BmNPV Bac-to-Bac快速表达系统的构建方法构建了HyNPV Bac-to-Bac快速表达系统。
本研究意义在于：(1)适用于家蚕BmNPV Bac-to-Bac快速系统的构建,在理论上突破了家蚕BmNPV表达系统中传统的重组病毒必须在昆虫细胞或昆虫活体内产生的思路,利用细菌转座子的位点特异性转座作用原理,在细菌内即可完成家蚕BmNPV重组病毒的构建,不仅在技术上有创新,而且丰富了家蚕杆状病毒表达系统的理论；在应用上提供了一个全新的技术平台,由于本研究建立的技术具有快速简便这一突出优点,很好地解决了目前家蚕杆状病毒表达系统应用中存在的技术繁琐、花费时间长这一长期困扰的关键问题,因此具有很好的应用前景。(2) BmNPV Bac-to-Bac (polh+)表达系统的构建,使重组病毒表型从芽生型病毒变为多角体病毒,该重组病毒能够经口接种家蚕,有效解决了一直困扰家蚕表达系统大规模应用中的重组病毒必须经皮接种这一瓶颈问题。(3)构建的HyNPV Bac-to-Bac(CP-)系统,较好解决了重组产物后期受蛋白酶降解的问题,有效提高了家蚕表达系统重组蛋白的生产效率。
Baculovirus has been proven to be the most powerful and versatile eukaryotic expression vectors available today. In this system, recombinant protein was expressed through the infection of insect or its culuture cells by using a recombinant baculovirus, which carrying the gene of interest. BEVS has lots of advantages including well high-level expression under control of strong promoter such as polyhedrin and p10 promoter, post-translational modifications, capacity of large gene insertions, as well as simultaneous expression of multiple genes. Compared with the mammalian expression, protein expressed by BEVS is safer to human being because the baculoviruses have very restricted host range.
The most popular vector in BEVS is Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). AcMNPV expression system has been well developed in many fields and was widely applied, specially in American and European countries. Compared with the AcMNPV system, the BmNPV was not applied as widely as AcMNPV, however, the most attractive advantage of BmNPV expression is that BmNPV can infect silkworm larva or pupa, which can be easily reared. When expressed in the silkworm, the expression can uauslly reach a higher level over other systems, moreover, the production cost is much lower. In our country, silkworm is very important economic insect, and large amount of the larvae can be easily obtained, and thus it is very promising to use the silkworm as a "biofactory" to produce the recombinant protein in biotechnological industry.
However, the BmNPV expression system has some bottlenecks in techniques, particularly in large-scale production. It includes:(1) The construction and purification of recombinant BmNPV is still laborious and time-taking. The traditional method for construction of recombinant BmNPV through homologous recombination in insect cells needs a few rounds of plaque assay. It needs skillful technique and usually takes 2-3 months; (2) In the late stage of infection, recombinant protein is usually degraded, which greatly decreases the protein production level. BEVS is a lytic expression system. There are several proteinase coded by the BmNPV itself. Along with the expression of foreign protein, the proteinases are also expressed, especially in the late stage of infection; (3) The efficiency of virus inoculation to the silkworm through subcutaneous injection is too low. Usually the polyhedrin gene in BmNPV was replaced by the foreign genes, so the recombinant BmNPV are polyhedrin negative virus. Without the protection of polyhedra, budded virus can hardly infect the silkworm by oral. The only way of recombinant BmNPV to infect silkworm larvae is through subcutaneous injection. For large-scale production, it is big problem.
This study tried to solve the above problems in order to make the BmNPV system more efficient. The achievements were summaried as the following:[1] A novel BmNPV Bac-to-Bac/Bacmid expression system suitable for silkworm, Bombyx mori was successfully developed. According to the principle of AcMNPV Bac-to-Bac expression system, the BmNPV genomic DNA was similarly reconstructed. A fragment containing a kanamycin resistance gene, a mini F replication element and the LacZ-αgene fragment which harbors a mini-attTn7 element as the target site of transposon without disrupting the LacZ open reading frame was inserted to the polyhedra site of BmNPV and replaced the polh gene through homologous recombination. Thus a BmNPV shuttle vector BmBacmid was constructed. Further, a set of donor plasmids essential for this system was reconstructed, and these donor plasmids make the recombinant protein well secreted. Using the newly constructed BmNPV Bac-to-Bac/Bacmid expression system, the recombinant BmNPV with foreign gene can be rapidly generated through bacterial transposition in E.coli. [2] A novel BmNPV expression system which can produce the polyhedrin was constructed. Polh gene was cloned to the site of p10 gene and placed in the control of p10 promoter. Thus the Bmbacmid (polh+) DNA were constructed. After the Bmbacmid (polh+) DNA and helper plamid were transformed into DH10βby turns, a recombinant polh+BmNPV carrying foreign gene can be rapidly constructed using Bac-to-Bac system. [3] Expression efficiency was increased through knockout of a main proteinase gene coded by BmNPV. The cyctein proteinase (CP) gene was knocked out of the HyNPV (a host-expanded BmNPV) through homologous recombinatoin.
Further, besides the BmNPV Bac-to-Bac system, HyNPV Bac-to-Bac expression system was also successfully established. The Hybacmid was constructed through replacement of polh gene by the above mentioned Kan-miniF-(attTn7)lacZ fragment.
This study has big scientific significance. It lies in (1) It offered a novel method to generate recombinant BmNPV by site-specific transposon in E.coli, challenging the traditional idea that recombinant BmNPV virus must be generated in insect larva or cultured insect cells. The BmNPV Bac-to-Bac system avoided the tedious, time-consuming plaque assay, so this new system was more efficient and shows good prospect in application. It provides a novel technical platform. (2) The BmNPV Bac-to-Bac (polh+) expression system can realize the oral infection. (3) The foreign protein production could be highly increased through reduction of protein degradation caused by proteinase.
目前昆虫杆状病毒表达系统因使用的病毒载体不同,主要可以分为两类,一类以苜蓿银纹夜蛾多核衣壳核型多角体病毒(Autographa californica multinucleocapsid nucleopolyhedrovirus,简写为AcMNPV)作为表达载体；另一类以家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,简写为BmNPV)作为表达载体。AcMNPV表达载体系统已经发展比较成熟,在欧美国家应用较多。与之不同,家蚕BmNPV由于可以用家蚕幼虫或蛹作为载体进行大规模的基因表达,蛋白质翻译后加工修饰完善,生产成本较低。特别在我国,家蚕是一种非常重要的特色经济资源昆虫,饲养技术非常成熟,在将来的基因工程产业化方面已显示出广阔的应用前景。
然而,家蚕BmNPV杆状病毒表达系统在技术上还存在许多欠缺,特别在进行大规模产业化开发方面还有一些技术瓶颈问题迫切需要解决,主要有以下几个方面：(1)重组病毒的构建过程繁琐,花费时间长。传统的重组BmNPV的构建和筛选方法还依赖于培养细胞内的基因同源重组和空斑分析,需要非常熟练的技巧,而且花费时间较长,通常需要2-3个月：(2)重组蛋白产物在后期常被降解,生产效率降低。杆状病毒表达系统是一个裂解性的系统,杆状病毒本身具有多个蛋白酶基因,在表达目的基因产物的同时,这些蛋白酶也得到了表达。特别是在感染晚期,重组蛋白易受蛋白酶降解,而感染的家蚕体内的蛋白质降解问题尤其严重；(3)目前感染接种采用的经皮接种技术,效率低下。为了达到较高的表达水平,目前构建重组病毒通常将目的基因替换多角体基因,从而形成不产生多角体的芽生型“裸露”重组病毒。重组病毒经口感染效率很低,因此常采用皮下注射感染的方法,不仅工效低,而且容易产生污染。
针对上述问题,本研究通过现代分子生物学技术和手段,借鉴国外经验,对家蚕杆状病毒表达系统进行了有力改造,使之成为一个更加高效的、能够适合产业化生产的表达系统。主要研究成果如下：[1]构建了适用于家蚕的BmNPV Bac-to-Bac/Bacmid快速高效表达系统。借鉴国外业已开发成功的AcMNPVBac-to-Bac快速基因表达系统工作原理,对家蚕BmNPV基因组进行了改造。通过同源重组的方法,将含有单拷贝数细菌F复制子、插入有细菌转座子整合靶位点、编码LacZa肽的部分DNA片段和抗性选择标记基因的基因片段重组入家蚕BmNPV基因组,替换多角体蛋白基因,获得了家蚕BmNPV病毒穿梭载体BmBacmid;并利用供体质粒上的表达盒和细菌转座子,和细菌转座子的基因定位转移作用,在细菌体内实现外源目的基因向家蚕BmNPV基因组上的转移整合,快速完成重组BmNPV病毒的构建。同时,对配套供体质粒进行了改造,构建带有分泌信号的供体质粒pBacGP67。[2]构建了能够表达多角体的重组BmNPV杆状病毒表达系统。将polh基因插入到p10位点,使用p10启动子控制多角体蛋白的表达。将筛选得到的p10被polh替换的Bmbacmid (polh+) DNA和helper质粒先后转化入DH10β,做成感受态细胞,得到同时含有Bmbacmid (polh+)和helper的DH10β,命名为DH10BmBac(polh+),结合上面构建的分泌型供体质粒,即构建成BmNPV Bac-to-Bac(polh+)快速基因表达系统。[3]删除了BmNPV杆状病毒自身的一种主要的蛋白酶基因,有效提高了表达效率。对杆状病毒基因组中引起蛋白降解的主要基因,半胱氨酸蛋白酶基因(CP),进行了基因敲除。同时,为了充分发挥本实验室构建的HyNPV病毒的优势,我们将Bac-to-Bac系统中的关键的8.6 kb片段按照BmNPV Bac-to-Bac快速表达系统的构建方法构建了HyNPV Bac-to-Bac快速表达系统。
本研究意义在于：(1)适用于家蚕BmNPV Bac-to-Bac快速系统的构建,在理论上突破了家蚕BmNPV表达系统中传统的重组病毒必须在昆虫细胞或昆虫活体内产生的思路,利用细菌转座子的位点特异性转座作用原理,在细菌内即可完成家蚕BmNPV重组病毒的构建,不仅在技术上有创新,而且丰富了家蚕杆状病毒表达系统的理论；在应用上提供了一个全新的技术平台,由于本研究建立的技术具有快速简便这一突出优点,很好地解决了目前家蚕杆状病毒表达系统应用中存在的技术繁琐、花费时间长这一长期困扰的关键问题,因此具有很好的应用前景。(2) BmNPV Bac-to-Bac (polh+)表达系统的构建,使重组病毒表型从芽生型病毒变为多角体病毒,该重组病毒能够经口接种家蚕,有效解决了一直困扰家蚕表达系统大规模应用中的重组病毒必须经皮接种这一瓶颈问题。(3)构建的HyNPV Bac-to-Bac(CP-)系统,较好解决了重组产物后期受蛋白酶降解的问题,有效提高了家蚕表达系统重组蛋白的生产效率。
Baculovirus has been proven to be the most powerful and versatile eukaryotic expression vectors available today. In this system, recombinant protein was expressed through the infection of insect or its culuture cells by using a recombinant baculovirus, which carrying the gene of interest. BEVS has lots of advantages including well high-level expression under control of strong promoter such as polyhedrin and p10 promoter, post-translational modifications, capacity of large gene insertions, as well as simultaneous expression of multiple genes. Compared with the mammalian expression, protein expressed by BEVS is safer to human being because the baculoviruses have very restricted host range.
The most popular vector in BEVS is Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). AcMNPV expression system has been well developed in many fields and was widely applied, specially in American and European countries. Compared with the AcMNPV system, the BmNPV was not applied as widely as AcMNPV, however, the most attractive advantage of BmNPV expression is that BmNPV can infect silkworm larva or pupa, which can be easily reared. When expressed in the silkworm, the expression can uauslly reach a higher level over other systems, moreover, the production cost is much lower. In our country, silkworm is very important economic insect, and large amount of the larvae can be easily obtained, and thus it is very promising to use the silkworm as a "biofactory" to produce the recombinant protein in biotechnological industry.
However, the BmNPV expression system has some bottlenecks in techniques, particularly in large-scale production. It includes:(1) The construction and purification of recombinant BmNPV is still laborious and time-taking. The traditional method for construction of recombinant BmNPV through homologous recombination in insect cells needs a few rounds of plaque assay. It needs skillful technique and usually takes 2-3 months; (2) In the late stage of infection, recombinant protein is usually degraded, which greatly decreases the protein production level. BEVS is a lytic expression system. There are several proteinase coded by the BmNPV itself. Along with the expression of foreign protein, the proteinases are also expressed, especially in the late stage of infection; (3) The efficiency of virus inoculation to the silkworm through subcutaneous injection is too low. Usually the polyhedrin gene in BmNPV was replaced by the foreign genes, so the recombinant BmNPV are polyhedrin negative virus. Without the protection of polyhedra, budded virus can hardly infect the silkworm by oral. The only way of recombinant BmNPV to infect silkworm larvae is through subcutaneous injection. For large-scale production, it is big problem.
This study tried to solve the above problems in order to make the BmNPV system more efficient. The achievements were summaried as the following:[1] A novel BmNPV Bac-to-Bac/Bacmid expression system suitable for silkworm, Bombyx mori was successfully developed. According to the principle of AcMNPV Bac-to-Bac expression system, the BmNPV genomic DNA was similarly reconstructed. A fragment containing a kanamycin resistance gene, a mini F replication element and the LacZ-αgene fragment which harbors a mini-attTn7 element as the target site of transposon without disrupting the LacZ open reading frame was inserted to the polyhedra site of BmNPV and replaced the polh gene through homologous recombination. Thus a BmNPV shuttle vector BmBacmid was constructed. Further, a set of donor plasmids essential for this system was reconstructed, and these donor plasmids make the recombinant protein well secreted. Using the newly constructed BmNPV Bac-to-Bac/Bacmid expression system, the recombinant BmNPV with foreign gene can be rapidly generated through bacterial transposition in E.coli. [2] A novel BmNPV expression system which can produce the polyhedrin was constructed. Polh gene was cloned to the site of p10 gene and placed in the control of p10 promoter. Thus the Bmbacmid (polh+) DNA were constructed. After the Bmbacmid (polh+) DNA and helper plamid were transformed into DH10βby turns, a recombinant polh+BmNPV carrying foreign gene can be rapidly constructed using Bac-to-Bac system. [3] Expression efficiency was increased through knockout of a main proteinase gene coded by BmNPV. The cyctein proteinase (CP) gene was knocked out of the HyNPV (a host-expanded BmNPV) through homologous recombinatoin.
Further, besides the BmNPV Bac-to-Bac system, HyNPV Bac-to-Bac expression system was also successfully established. The Hybacmid was constructed through replacement of polh gene by the above mentioned Kan-miniF-(attTn7)lacZ fragment.
This study has big scientific significance. It lies in (1) It offered a novel method to generate recombinant BmNPV by site-specific transposon in E.coli, challenging the traditional idea that recombinant BmNPV virus must be generated in insect larva or cultured insect cells. The BmNPV Bac-to-Bac system avoided the tedious, time-consuming plaque assay, so this new system was more efficient and shows good prospect in application. It provides a novel technical platform. (2) The BmNPV Bac-to-Bac (polh+) expression system can realize the oral infection. (3) The foreign protein production could be highly increased through reduction of protein degradation caused by proteinase.
引文
[1]Patterson RM, Selkirk JK, Merrick BA. Baculovirus and insect cell gene expression-review of baculovirus biotechnology. Environ Health Perspect,1995,103:756-759.
[2]Beljelarskaya SN. A baculovirus expression system for insect cells. Mol Biol,2002,36: 281-292.
[3]Francki RIB, Fanquet CM, Knudson DL, et al. Classification and nomenclature of viruses: Fifth Report of the International Committee on Taxonomy of Viruses. Arch Virol, 1991(Suppl),2:1-450.
[4]Horton HM, Burand JM. Saturable attachment sites for polyhedron-derived baculovirus on insect cells and evidence for entry via direct membrane fusion. J Virol,1993,67(4): 1860-1868.
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