VP60蛋白的原核表达、抗体制备及VP60基因转化的研究
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摘要
兔出血症(rabbit hemorrhagic disease,RHD)是由兔出血症病毒（rabbit hemorrhagicdisease virus，RHDV）引起的兔的一种疾病。该病具有高度传染，高发病率、高致死性的特点。成年兔染病后以全身实质器官水肿、淤血及出血性变化为主要特征，其发病率和致死率都很高，是兔的一种毁灭性传染病。兔出血症病毒（RHDV）属于杯状病毒科（Caliciviridae），兔病毒属（Lagovirus）。RHDV基因组为单股正链RNA，全长7437bp，基因组含两个开放阅读框(open reading frame,ORF)，即ORF1和ORF2，ORF1编码一个含有2344个氨基酸残基的多聚蛋白(257kD)，C末端包含衣壳蛋白VP60；ORF2位于基因组的3’端，编码一个小衣壳蛋白VP10。VP60蛋白的分子质量约为60kD，故称为VP60，是RHDV的主要结构蛋白，它在诱导抗病毒感染的免疫反应中起重要作用，是病毒免疫保护性抗原。目前使用的RHDV疫苗是用染病兔子肝脏提取物作为组织灭活苗，或者用原核表达的重组VP60蛋白作为抗原免疫兔子，以预防兔出血症疾病。这些方式虽然具有良好的免疫效果，但也有自身所不可避免的种种缺点。近些年，随着植物基因工程技术的发展，通过转基因植物生产疫苗成为现实。所以，当前RHDV疫苗的研究主要采用植物基因工程技术进行疫苗的研制。
本研究在克隆了RHDVVP60基因的基础上对VP60的原核表达抗体制备进行研究，利用农杆菌转化法将VP60基因进行转化烟草和百脉根，同时对苜蓿再生体系的建立进行了探索，取得以下结果：
1.构建兔出血症病毒衣壳蛋白VP60基因的原核表达载体，进行原核表达并制备其蛋白抗体。以实验室获得并保存VP60基因的质粒(pGEM-Teasy-vp60)为模版，采用PCR方法获得VP60基因，将其克隆到pET28a(+)载体上，构建重组表达载体pET28a(+)-vp60，酶切鉴定和测序验证后转入大肠杆菌BL21(DE3)中进行诱导表达，用0.4mmol/LIPTG在20℃的条件下诱导8h，VP60蛋白得到了高效表达，利用镍离子螯合层析纯化重组蛋白，注射兔子后制备了抗VP60多克隆抗体，制备的多克隆抗体具有较强的免疫结合活性。为进一步研究VP60转基因奠定了基础。
2.以烟草SR1叶片为外植体材料，用构建好的含有拟南芥atslA基因启动子的植物转化载体pCAMBIA1300-ats1A-vp60转入根癌农杆菌EHA105中，然后对烟草叶片进行根癌农杆菌介导的转化研究。结果表明，转化的外植体叶片在MS+6-BA3mg/L培养基上28℃共培养2d后，转入含有潮霉素21mg/L培养基上筛选，再生芽生根后，提取转化植株基因组DNA，进行PCR检测和Southernblot检测，有部分转化植株中整合了目的基因；进一步的提取转基因植株的RNA，反转录PCR结果表明，植株中含有整合的目的基因，Westernblot检测初步证实有微量的目的蛋白在烟草中表达。
3.建立了百脉根的高频再生体系，百脉根在MS+6-BA0.2mg/L培养基上下胚轴再生率可达到100%，外植体下胚轴再生率明显比子叶的再生率高，根再生的最优培养基为MS+6-BA0.2mg/L；构建了pCAMBIA1301-vp60基因植物表达载体，确立了百脉根最适潮霉素筛选浓度为2.0mg/L，并利用根癌农杆菌EHA105（含pCAMBIA1301-vp60）介导对百脉根的下胚轴进行遗传转化。转化后经过潮霉素筛选，再生芽转入根再生培养基（含潮霉素）上得到转化植株，提取转化植株的基因组DNA及RNA，基因组PCR检测和反转录PCR检测的结果表明部分的转化植株含有目的基因VP60。
4.对苜蓿金皇后的外植体下胚轴进行了再生体系的研究，最佳的消毒条件是75%酒精消毒1min，0.1%升汞消毒5min；对不同的激素配比下分别进行愈伤组织诱导的筛选，确定最佳的培养基为MS+NAA1.8mg/L+6-BA1.6mg/L；利用不同的激素诱导愈伤组织分化，最佳的诱导培养基配比为MS+NAA0.3mg/L+6-BA0.6mg/L。用诱导分化幼苗下胚轴进行潮霉素筛选，不同浓度的潮霉素对紫花苜蓿诱导愈伤结果表明，各浓度对愈伤组织的分化均有一定的抑制作用，且随浓度升高，抑制作用增强，确定最适的选择压力浓度为15mg/L。为下一步VP60基因转化苜蓿创制新型牧草奠定了基础。
Rabbit hemorrhagic disease (RHD) is caused by the Rabbit Hemorrhagic Disease (RHDV). It is a highly contagious and acute disease with high morbidity and mortality in adult rabbits. The disease is characterized by severe liver damage in combination with a disseminated intravascular coagulation syndrome leading to hemorrhaging in different organs of adult rabbit. The RHDV genome is a single-stranded, positive-sense RNA molecule,7,437nt in length. Sequencing of the genome has revealed the presence of two open reading frames (ORFs). ORF1encodes a polyprotein of257kDa and includes the capsid protein, VP60, at its C-terminus. ORF2is located at the3'end of the genome and encodes a small capsid protein, VP10. The VP60capsid protein, whose molecular size is about60kDa, has various functions, including assembly into the capsid, as well as interaction with host proteins to mediate host-cell receptor binding and antigenic diversity, conferring host specificity and antigenicity. At present, there are several available vaccines against rabbit hemorrhagic disease on the market, which is the main component of commercially used vaccines isolated from liver extracts of infected rabbit or recombinant protein VP60expressed in several heterologous systems like Escherichia coli, Saccaromyces cerevisiae and so on. Although they have proved to be effective tools for prevention of the disease, there are many problems resulting from the use of infectious virus and the risk of its dissemination from vaccine factories. Thus, it is important to develop alternative approaches for producing vaccines. In recent years, transgenic plants have demonstrated considerable potential for the production of vaccines. Plants are a promising platform for the production of vaccines.
This investigation, based on the clone of vp60gene of RHDV, the expression of recombinant protein VP60in Escherichia coli and preparation of its polyclonal antibody were studied, tobacco and Lotus corniculatus L. were transformed by Agrobacterium tumefaciens with the gene vp60, and also the regeneration of alfalfa (Medicago sativa. Golden Empress) was explored. The major results are as follows:
1. The rabbit hemorrhagic disease virus (RHDV) capsid protein (VP60) gene was obtained by PCR, and cloned into prokaryotic expression vector pET28a(+). The identified construct was transformed into E. coli and over-expressed protein was purified by nickel chelating chromatography and polyclonal antiserum was raised against rabbit. The vp60gene was amplified by PCR. The recombinant prokaryotic expression vector pET28a(+)-vp60was constructed by molecular technique, and then the recombinant was transformed into E. coli BL21(DE3) to induce protein expression with IPTG. The recombinant protein purified by nickel chelating chromatography was used as antigen to immunize rabbit for preparation of polyclonal antibody. The protein VP60polyclonal antibody has provided reliable tools for future study in the transgenic plant of vp60.
2. The VP60gene from RHDV (rabbit hemorrhagic disease virus) YL Strain in Northeast of China, under control of the ast1A promoter from Rubisco small subunite genes of Arabidopsis thaliana, was introduced into the T-DNA region of plant transfer vector pCAMBIA1300and transferred to tobacco(Nicotiana tabacum cv. Petit Havanna SR1)with Agrobacterium tumefaciens-mediated method. PCR and RT-PCR analysis of the transformed tobacco plants confirmed the integration of the vp60gene copy into the plant DNA and vp60gene transcription produced. Western blot analysis revealed that the VP60protein was expressed in tobacco under control of ast1A promoter.
3. The regeneration system of Lotus corniculatus L. was established. On the medium MS+6-BA0.2mg/L, the hypocotyls and the cotyledon from Lotus corniculatus L. were induced easily and directly to be buds with regeneration rate100%and93.3%respectively. The regeneration plants were rooted on the medium MS0or MS0+NAA0.1mg/L. The vp60gene from RHDV (rabbit hemorrhagic disease virus) YL Strain in Northeast of China was introduced into the T-DNA region of plant transfer vector pCAMBIA1301transferred to hypocotyls of Lotus corniculatus L. Agrobacterium tumefaciens-mediated method. PCR and RT-PCR analysis of the transformed Lotus corniculatus L. plants which grew on the medium MS0+6-BA0.2mg/L+hyg2mg/L, confirmed the integration of the vp60gene copy into the plant DNA and vp60gene transcription produced.
4. The alfalfa (Golden Empress) was studied on establishing the regeneration system. The best time for the disinfection was lmin in75%alcohol, then5min in0.1%mercuric chloride. The experiment studied the effects of different hormones variety and ratio for the alfalfa on the callus induction and induced differentiation. The optimal medium for callus differentiation was MS+NAA1.8mg/L+6-BA1.6mg/L, the optimal medium for bud differentiation was MS+6-BA0.6mg/L+NAA0.3mg/L. Induced differentiation of hypocotyls of seedlings were put on the mediums including different concentration of hygromycin for optimal screening. Different concentrations of hygromycin had some restraining effects on callus inducement and differentiation in alfalfa hypocotyl, and the restraining effect enhanced along with the rising concentration of hygromycin, and the concentration for selecting was15mg/L.
本研究在克隆了RHDVVP60基因的基础上对VP60的原核表达抗体制备进行研究，利用农杆菌转化法将VP60基因进行转化烟草和百脉根，同时对苜蓿再生体系的建立进行了探索，取得以下结果：
1.构建兔出血症病毒衣壳蛋白VP60基因的原核表达载体，进行原核表达并制备其蛋白抗体。以实验室获得并保存VP60基因的质粒(pGEM-Teasy-vp60)为模版，采用PCR方法获得VP60基因，将其克隆到pET28a(+)载体上，构建重组表达载体pET28a(+)-vp60，酶切鉴定和测序验证后转入大肠杆菌BL21(DE3)中进行诱导表达，用0.4mmol/LIPTG在20℃的条件下诱导8h，VP60蛋白得到了高效表达，利用镍离子螯合层析纯化重组蛋白，注射兔子后制备了抗VP60多克隆抗体，制备的多克隆抗体具有较强的免疫结合活性。为进一步研究VP60转基因奠定了基础。
2.以烟草SR1叶片为外植体材料，用构建好的含有拟南芥atslA基因启动子的植物转化载体pCAMBIA1300-ats1A-vp60转入根癌农杆菌EHA105中，然后对烟草叶片进行根癌农杆菌介导的转化研究。结果表明，转化的外植体叶片在MS+6-BA3mg/L培养基上28℃共培养2d后，转入含有潮霉素21mg/L培养基上筛选，再生芽生根后，提取转化植株基因组DNA，进行PCR检测和Southernblot检测，有部分转化植株中整合了目的基因；进一步的提取转基因植株的RNA，反转录PCR结果表明，植株中含有整合的目的基因，Westernblot检测初步证实有微量的目的蛋白在烟草中表达。
3.建立了百脉根的高频再生体系，百脉根在MS+6-BA0.2mg/L培养基上下胚轴再生率可达到100%，外植体下胚轴再生率明显比子叶的再生率高，根再生的最优培养基为MS+6-BA0.2mg/L；构建了pCAMBIA1301-vp60基因植物表达载体，确立了百脉根最适潮霉素筛选浓度为2.0mg/L，并利用根癌农杆菌EHA105（含pCAMBIA1301-vp60）介导对百脉根的下胚轴进行遗传转化。转化后经过潮霉素筛选，再生芽转入根再生培养基（含潮霉素）上得到转化植株，提取转化植株的基因组DNA及RNA，基因组PCR检测和反转录PCR检测的结果表明部分的转化植株含有目的基因VP60。
4.对苜蓿金皇后的外植体下胚轴进行了再生体系的研究，最佳的消毒条件是75%酒精消毒1min，0.1%升汞消毒5min；对不同的激素配比下分别进行愈伤组织诱导的筛选，确定最佳的培养基为MS+NAA1.8mg/L+6-BA1.6mg/L；利用不同的激素诱导愈伤组织分化，最佳的诱导培养基配比为MS+NAA0.3mg/L+6-BA0.6mg/L。用诱导分化幼苗下胚轴进行潮霉素筛选，不同浓度的潮霉素对紫花苜蓿诱导愈伤结果表明，各浓度对愈伤组织的分化均有一定的抑制作用，且随浓度升高，抑制作用增强，确定最适的选择压力浓度为15mg/L。为下一步VP60基因转化苜蓿创制新型牧草奠定了基础。
Rabbit hemorrhagic disease (RHD) is caused by the Rabbit Hemorrhagic Disease (RHDV). It is a highly contagious and acute disease with high morbidity and mortality in adult rabbits. The disease is characterized by severe liver damage in combination with a disseminated intravascular coagulation syndrome leading to hemorrhaging in different organs of adult rabbit. The RHDV genome is a single-stranded, positive-sense RNA molecule,7,437nt in length. Sequencing of the genome has revealed the presence of two open reading frames (ORFs). ORF1encodes a polyprotein of257kDa and includes the capsid protein, VP60, at its C-terminus. ORF2is located at the3'end of the genome and encodes a small capsid protein, VP10. The VP60capsid protein, whose molecular size is about60kDa, has various functions, including assembly into the capsid, as well as interaction with host proteins to mediate host-cell receptor binding and antigenic diversity, conferring host specificity and antigenicity. At present, there are several available vaccines against rabbit hemorrhagic disease on the market, which is the main component of commercially used vaccines isolated from liver extracts of infected rabbit or recombinant protein VP60expressed in several heterologous systems like Escherichia coli, Saccaromyces cerevisiae and so on. Although they have proved to be effective tools for prevention of the disease, there are many problems resulting from the use of infectious virus and the risk of its dissemination from vaccine factories. Thus, it is important to develop alternative approaches for producing vaccines. In recent years, transgenic plants have demonstrated considerable potential for the production of vaccines. Plants are a promising platform for the production of vaccines.
This investigation, based on the clone of vp60gene of RHDV, the expression of recombinant protein VP60in Escherichia coli and preparation of its polyclonal antibody were studied, tobacco and Lotus corniculatus L. were transformed by Agrobacterium tumefaciens with the gene vp60, and also the regeneration of alfalfa (Medicago sativa. Golden Empress) was explored. The major results are as follows:
1. The rabbit hemorrhagic disease virus (RHDV) capsid protein (VP60) gene was obtained by PCR, and cloned into prokaryotic expression vector pET28a(+). The identified construct was transformed into E. coli and over-expressed protein was purified by nickel chelating chromatography and polyclonal antiserum was raised against rabbit. The vp60gene was amplified by PCR. The recombinant prokaryotic expression vector pET28a(+)-vp60was constructed by molecular technique, and then the recombinant was transformed into E. coli BL21(DE3) to induce protein expression with IPTG. The recombinant protein purified by nickel chelating chromatography was used as antigen to immunize rabbit for preparation of polyclonal antibody. The protein VP60polyclonal antibody has provided reliable tools for future study in the transgenic plant of vp60.
2. The VP60gene from RHDV (rabbit hemorrhagic disease virus) YL Strain in Northeast of China, under control of the ast1A promoter from Rubisco small subunite genes of Arabidopsis thaliana, was introduced into the T-DNA region of plant transfer vector pCAMBIA1300and transferred to tobacco(Nicotiana tabacum cv. Petit Havanna SR1)with Agrobacterium tumefaciens-mediated method. PCR and RT-PCR analysis of the transformed tobacco plants confirmed the integration of the vp60gene copy into the plant DNA and vp60gene transcription produced. Western blot analysis revealed that the VP60protein was expressed in tobacco under control of ast1A promoter.
3. The regeneration system of Lotus corniculatus L. was established. On the medium MS+6-BA0.2mg/L, the hypocotyls and the cotyledon from Lotus corniculatus L. were induced easily and directly to be buds with regeneration rate100%and93.3%respectively. The regeneration plants were rooted on the medium MS0or MS0+NAA0.1mg/L. The vp60gene from RHDV (rabbit hemorrhagic disease virus) YL Strain in Northeast of China was introduced into the T-DNA region of plant transfer vector pCAMBIA1301transferred to hypocotyls of Lotus corniculatus L. Agrobacterium tumefaciens-mediated method. PCR and RT-PCR analysis of the transformed Lotus corniculatus L. plants which grew on the medium MS0+6-BA0.2mg/L+hyg2mg/L, confirmed the integration of the vp60gene copy into the plant DNA and vp60gene transcription produced.
4. The alfalfa (Golden Empress) was studied on establishing the regeneration system. The best time for the disinfection was lmin in75%alcohol, then5min in0.1%mercuric chloride. The experiment studied the effects of different hormones variety and ratio for the alfalfa on the callus induction and induced differentiation. The optimal medium for callus differentiation was MS+NAA1.8mg/L+6-BA1.6mg/L, the optimal medium for bud differentiation was MS+6-BA0.6mg/L+NAA0.3mg/L. Induced differentiation of hypocotyls of seedlings were put on the mediums including different concentration of hygromycin for optimal screening. Different concentrations of hygromycin had some restraining effects on callus inducement and differentiation in alfalfa hypocotyl, and the restraining effect enhanced along with the rising concentration of hygromycin, and the concentration for selecting was15mg/L.
引文
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