一种新型的抗血管紧张素II的治疗性高血压疫苗的构建、表达及治疗效果的研究
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摘要
高血压病(Hypertension, HTN)是一种以动脉血压持续偏高为主要表现的慢性疾病,常伴有脂肪和糖代谢紊乱以及心、脑、肾和视网膜等器官组织的不良重塑,其脑卒中、心肌梗死、心力衰竭及慢性肾脏病等主要并发症,致残、致死率高。据报道,目前全球约有10亿高血压患者,每年有近800万人死于由高血压引起的各种疾病,我国有高血压患者至少2亿,每年新增患者约1000万人。目前治疗高血压主要是通过药物治疗,包括α-受体阻滞剂类,钙通道阻断剂类,血管紧张素转化酶抑制剂类,血管紧张素Ⅱ受体拮抗剂类等。虽然药物治疗疗效明显,但由于患者服药依从性差,药物引起的毒副作用以及机体产生耐药性等原因,导致目前高血压控制率不高,因此世界各国心血管基础研究和临床工作者都在积极探索新的治疗方法和手段,而开发高效、安全的疫苗成为治疗和控制高血压病的一个新的研究方向。
肾素-血管紧张素-醛固酮系统(The renin-angiotensin-aldosteron system, RAAS)过度激活是目前研究的最为清楚,也是最为重要的高血压发病机制,在RAAS中,过多的血管紧张素Ⅱ(AngiotensinⅡ, AngⅡ)通过与主要存在于血管,以及心、脑、肾的血管紧张素Ⅱ受体Ⅰ型(AngⅡ type Ⅰ receptor, ATIR)结合,引起血管强烈收缩等一系列的生理效应,导致血压升高。AngⅡ是迄今为止最强的收缩血管的活性物质之一。如果机体内存在抗AngⅡ的特异性抗体,能与过多的AngⅡ发生中和反应,使之维持在正常水平,就能达到降低血压的效果。本课题就是以AngⅡ作为靶点来探索研究一种新型的治疗性高血压疫苗,该疫苗是以4段连续AngⅡ肽段为核心,以甲型肝炎病毒类病毒颗粒(Hepatitis A virus-like particle, HAV-VLP)作为载体的嵌合蛋白,在免疫机体后,希望该疫苗能够刺激机体的免疫反应,产生抗AngⅡ的特异性抗体,达到降压效果。
在本课题中,我们首先通过基因工程手段合成4个连续重复的人源的AngⅡ基因序列(4AngⅡs),将其插入HAV基因组中,并利用重组PCR技术扩增该基因组上P1-2A-4AngⅡs和3ABC基因,将其分别克隆至杆状病毒表达系统中供体质粒pFastBac-Dual的PPH和PP10启动子下游的多克隆位点处,获得重组供体质粒,将该重组供体质粒转化E. coli DH10,并与E. coli DH10细胞中杆粒发生转座获得重组杆粒,用该重组杆粒转染昆虫细胞sf9系包装重组杆状病毒Bav-P1-2A-4AngIIs-3ABC。通过PCR,双酶切以及基因测序等方法对各重组质粒进行鉴定,通过观察细胞病变(CPE), PCR,基因测序以及透射电镜等方法对获得重组杆状病毒进行鉴定,并采用蚀斑实验对其病毒滴度进行测定。本课题中采用Bac-to-Bac杆状病毒双表达系统表达目的蛋白pHAV-4AngⅡs,通过RT-PCR, SDS-PAGE,间接免疫荧光,Western Blot,透射电镜等方法对该嵌合蛋白的表达进行鉴定,通过蔗糖密度梯度超速离心的方法对pHAV-4AngⅡs进行纯化,收获高浓度、高纯度的目的蛋白。PHAV-4AngⅡs免疫治疗自发性高血压大鼠(spontaneously hypertensive rats, SHRs)后,通过无创尾压测量法测量SHRs的血压,ELISA方法检测大鼠血清中抗AngⅡ特异性抗体滴度以及血清中AngⅡ含量,最后利用T检验和Pearson双变量相关性分析对各数据进行统计学分析,考察该疫苗的免疫原性及治疗效果。
本课题中成功包装并获得携带目的基因P1-2A-4AngⅡs和3ABC的重组杆状病毒Bav-P1-2A-4AngⅡs-3ABC表达载体,利用PCR方法对其进行验证,能特异性的扩增得到P1-2A-4AngIIs和3ABC基因片段,基因测序结果显示序列均与预期一致,读码框架正确。透射电镜下能观察到典型的杆状病毒颗粒结构,通过蚀斑实验测得P3代重组杆状病毒病毒滴度约为4×108pfu/ml。重组杆状病毒Bav-P1-2A-4AngⅡs-3ABC感染sf9细胞后,在sf9细胞中成功表达目的蛋白pHAV-4AngⅡs,通过RT-PCR能特异性的扩增得到P1-2A-4AngⅡs和3ABC基因片段,证实这两个基因片段在RNA水平发生转录；、Vestern blot结果显示在约Mr37000处有明显的蛋白杂交带,其大小正好与目的蛋白pHAV-4AngⅡs的亚基VP1-2A-4AngⅡs分子量一致；免疫荧光结果显示：接种重组杆状病毒的sf9细胞中出现特异性荧光,而未接种病毒的sf9细胞中则未发现特异性荧光；透射电镜下能够观察到pHAV-4AngⅡs的VLP结构,直径约22nm;通过ELISA和Western blot方法探索得到pHAV-4AngIIs的最佳表达条件：MOI=1,表达时间为96h。目的蛋白经蔗糖密度梯度超速离心后,免疫治疗SHRs,发现该疫苗能诱导机体产生较高滴度,持续时间约为10周的抗AngⅡ特异性1gG抗体,在第8周抗体滴度达到峰值时,pHAV-4AngⅡs组SHRs跟PBS组SHRs进行比较,平均收缩压降低约23mmHg,平均舒张压降低约12mmHg,血清中Angll的平均浓度降低约87pg/ml。利用Pearson双变量相关性分析对Angll浓度,血压,抗Angll抗体滴度三者进行统计学分析,结果显示：Angll浓度与血压之间存在正相关性；Angll浓度和抗Angll抗体滴度之间存在负相关性；血压和抗Angll抗体滴度存在负相关性。
本试验最终表明：以Angll为功能表位,HAV-VLP为载体的嵌合蛋白pHAV-4AngIIs具有很好的免疫原性和抗原性,可诱导机体产生较高滴度的抗AngⅡ的特异性抗体,该抗体能够有效中和血清中过多的AngⅡ,阻断RAAS,对SHRs具有一定的降压治疗效果。这为高血压疫苗的研究和开发提供了一种新的思路,具有广阔的应用前景。
Hypertension (HTN) is a chronic disease characterized by the persistent high arterial blood pressure, always accompanied the disorder of fat and carbohydrate metabolism, and accompanied the organs or tissues remodeling of heart, brain, kidneys and retina. High rates of disability and mortality are associated with HTN's complications, which include stroke, myocardial infarction, cardiac failure and chronic kidney disease. As reported, nearly one billion people currently have hypertension, which kills8million people every year worldwide. In china, there are two billion hypertensive patients at least, about10million new patients each year. Currently, hypertensive patients are treated with a blockers, calcium channel blockers (CCBs), ACE inhibitors (ACEIs) and angiotensin Ⅱ receptor antagonists. Although these medicines showed good therapeutic effect, hypertension cannot be effectively controlled due to their serious side-effects, the emergence of resistance to medicines and the poor compliance. So the cardiovascular researchers and clinical workers worldwide are actively exploring new treatments. The development of efficient, safe vaccine becomes a new research direction for the treatment and control of hypertension.
The renin-angiotensin-aldosteron system (RAAS) is the most important system involved in the onset of hypertension. AngⅡ plays an important role in this system; it causes a series of physiological effects by binding with the AngⅡ type I receptor (AT1R) on the blood vessels, heart, brain and kidneys to increase blood pressure. AngⅡ is one of the most powerful active substances to cause strong vasoconstriction and increase blood pressure. If there are anti-AngⅡ-specific antibodies in vivo, which can neutralize the serum excessive Angll and keep it on the normal level, blood pressure could be reduced. In this study, AngⅡ was selected as target to explore a novel anti-hypertension therapeutic vaccine. This vaccine is a chimeric protein which presents four successive repeated AngⅡs as the functional epitope on the surface of the hepatitis A virus-like particle (HAVLP) to elevate the immunogenicity of AngⅡ. We hope that the vaccine can activate the immune response of the host, induce the anti-AngⅡ specific neutralizing antibody to block the RAAS, and blood pressure subsequently can be decreased after immunizations.
In this study, four-artificial-repeated AngⅡ s DNA sequence was inserted into the2A region of plasmid pT7-18f obtained by modifying the HAV genome. The recombinant plasmid pT7-18f was used as template to amplify the gene P1-2A-4AngⅡs and the gene3ABC by PCR. These two gene fragments were inserted into the multiple clone sites located downstream of the PH and P10promoters, respectively, on pFastBacTM-dual vectors. The recombinant pFastBac-dual plasmids were transformed into DH10E.coli for transposition into the bacmid to obtain the recombinant bacmids. Then sf9cells were transfected with the recombinant bacmids using cellfectinⅡ reagent to obtain recombinant baculoviruses Bav-P1-2A-4AngIIs-3ABC. These recombinant plasmids above were identified by PCR, double enzyme digestion and gene sequencing methods. The recombinant baculoviruses were identified by observing cytopathic effect (CPE), PCR, gene sequencing and transmission electron microscope, and the plaque assay was used to determine the titer of recombinant baculoviruses. In this study, the target protein pHAV-4AngⅡs was expressed by Bac-to-Bac baculovirus expression system, identified through RT-PCR, SDS-PAGE, indirect immunofluorescence, western blot, transmission electron microscope and other methods, and purified by sucrose density gradient ultracentrifugation. Spontaneously hypertensive rats (SHRs) were immuned with the purified pHAV-4AngⅡs. Blood pressure in SHRs was measured by tail-cuff arterial blood pressure measurement method. The serum AngⅡ levels and the anti-AngⅡ specific antibody in sera were tested by ELISA. At last, these data were analysised though T test and Pearson bivariate correlation analysis to study immunogenicity and treatment effect of pHAV-4AngIIs.
In this study, the recombinant baculoviruses carrying P1-2A-4AngⅡs and3ABC genes were successfully constructed. It was identified using PCR, and P1-2A-4AngⅡs and3ABC gene fragments were specifically amplified. The result of gene sequencing showed that the sequences and the reading frames are correct. The typical baculovirus particles were observed under the transmission electron microscope, and the plaque assay suggested that titer of the P3viruses was approximately4×108pfu/ml. PHAV-4AngⅡs were successfully expressed in sf9cells which were infected the recombinant baculoviruses. Through RT-PCR, P1-2A-4AngIIs and3ABC genes were amplified using sf9cells infected with recombinant baculoviruses and could not be amplified using normal sf9cells. These findings suggested that these two genes can be successfully transcribed in infected sf9cells. Western blot exposed a specific protein band at a molecular mass of～37KD and the protein band may represent the VP1-2A-4AngⅡs subunit of pHAV-4AngⅡs. Specifically, fluorescence were observed in infected sf9cells, and no fluorescence were observed in normal cells, suggesting that pHAV-4AngⅡs were successfully expressed in the infected sf9cells, which provides further evidence of good immunoreactivity. The VLPs of pHAV-4AngIIs were observed and approximately22nm under transmission electron microscope. The results from ELISA and western blot suggested that the optimum expression of pHAV-4AngIIs:MOI=1, time=96h. SHRs were immunized with the purified pHAV-4AngIIs to test immunogenicity and pharmacodynamic action. The results showed that this vaccine can induce high titer anti-AngⅡ-specific IgG antibody for almost10weeks. When antibody titer reach the peak at8th week, compared the PBS group SHRs, the mean systolic blood pressure (SBP) on pHAV-4AngⅡs group SHRs degraded approximately23mmHg, the mean diastolic blood pressure (DBP) degraded approximately12mmHg, and the mean concentration of AngⅡ in sera degraded approximately87pg/ml. The results of Pearson bivariate correlation analysis showed that there is a direct correlation between AngⅡ level and blood pressure, an inverse correlation between AngⅡ level and anti-AngⅡ-specific antibody titer, an inverse correlation between blood pressure and anti-AngⅡ-specific antibody titer.
In conclusion, the chimeric protein pHAV-4AngIIs presenting AngⅡ as functional epitope on the surface of HAVLP have good immunogenicity and antigenicity, and also have good effect on reduction of blood pressure in SHRs. It can induce high titer of anti-AngⅡ-specific antibody which can neutralize·excessive AngⅡ in sera, block RAAS and lower blood pressure. These findings provide that the pHAV-4AngIIs would be a new direction of exploration for the development of anti-hypertension therapeutic vaccine.
肾素-血管紧张素-醛固酮系统(The renin-angiotensin-aldosteron system, RAAS)过度激活是目前研究的最为清楚,也是最为重要的高血压发病机制,在RAAS中,过多的血管紧张素Ⅱ(AngiotensinⅡ, AngⅡ)通过与主要存在于血管,以及心、脑、肾的血管紧张素Ⅱ受体Ⅰ型(AngⅡ type Ⅰ receptor, ATIR)结合,引起血管强烈收缩等一系列的生理效应,导致血压升高。AngⅡ是迄今为止最强的收缩血管的活性物质之一。如果机体内存在抗AngⅡ的特异性抗体,能与过多的AngⅡ发生中和反应,使之维持在正常水平,就能达到降低血压的效果。本课题就是以AngⅡ作为靶点来探索研究一种新型的治疗性高血压疫苗,该疫苗是以4段连续AngⅡ肽段为核心,以甲型肝炎病毒类病毒颗粒(Hepatitis A virus-like particle, HAV-VLP)作为载体的嵌合蛋白,在免疫机体后,希望该疫苗能够刺激机体的免疫反应,产生抗AngⅡ的特异性抗体,达到降压效果。
在本课题中,我们首先通过基因工程手段合成4个连续重复的人源的AngⅡ基因序列(4AngⅡs),将其插入HAV基因组中,并利用重组PCR技术扩增该基因组上P1-2A-4AngⅡs和3ABC基因,将其分别克隆至杆状病毒表达系统中供体质粒pFastBac-Dual的PPH和PP10启动子下游的多克隆位点处,获得重组供体质粒,将该重组供体质粒转化E. coli DH10,并与E. coli DH10细胞中杆粒发生转座获得重组杆粒,用该重组杆粒转染昆虫细胞sf9系包装重组杆状病毒Bav-P1-2A-4AngIIs-3ABC。通过PCR,双酶切以及基因测序等方法对各重组质粒进行鉴定,通过观察细胞病变(CPE), PCR,基因测序以及透射电镜等方法对获得重组杆状病毒进行鉴定,并采用蚀斑实验对其病毒滴度进行测定。本课题中采用Bac-to-Bac杆状病毒双表达系统表达目的蛋白pHAV-4AngⅡs,通过RT-PCR, SDS-PAGE,间接免疫荧光,Western Blot,透射电镜等方法对该嵌合蛋白的表达进行鉴定,通过蔗糖密度梯度超速离心的方法对pHAV-4AngⅡs进行纯化,收获高浓度、高纯度的目的蛋白。PHAV-4AngⅡs免疫治疗自发性高血压大鼠(spontaneously hypertensive rats, SHRs)后,通过无创尾压测量法测量SHRs的血压,ELISA方法检测大鼠血清中抗AngⅡ特异性抗体滴度以及血清中AngⅡ含量,最后利用T检验和Pearson双变量相关性分析对各数据进行统计学分析,考察该疫苗的免疫原性及治疗效果。
本课题中成功包装并获得携带目的基因P1-2A-4AngⅡs和3ABC的重组杆状病毒Bav-P1-2A-4AngⅡs-3ABC表达载体,利用PCR方法对其进行验证,能特异性的扩增得到P1-2A-4AngIIs和3ABC基因片段,基因测序结果显示序列均与预期一致,读码框架正确。透射电镜下能观察到典型的杆状病毒颗粒结构,通过蚀斑实验测得P3代重组杆状病毒病毒滴度约为4×108pfu/ml。重组杆状病毒Bav-P1-2A-4AngⅡs-3ABC感染sf9细胞后,在sf9细胞中成功表达目的蛋白pHAV-4AngⅡs,通过RT-PCR能特异性的扩增得到P1-2A-4AngⅡs和3ABC基因片段,证实这两个基因片段在RNA水平发生转录；、Vestern blot结果显示在约Mr37000处有明显的蛋白杂交带,其大小正好与目的蛋白pHAV-4AngⅡs的亚基VP1-2A-4AngⅡs分子量一致；免疫荧光结果显示：接种重组杆状病毒的sf9细胞中出现特异性荧光,而未接种病毒的sf9细胞中则未发现特异性荧光；透射电镜下能够观察到pHAV-4AngⅡs的VLP结构,直径约22nm;通过ELISA和Western blot方法探索得到pHAV-4AngIIs的最佳表达条件：MOI=1,表达时间为96h。目的蛋白经蔗糖密度梯度超速离心后,免疫治疗SHRs,发现该疫苗能诱导机体产生较高滴度,持续时间约为10周的抗AngⅡ特异性1gG抗体,在第8周抗体滴度达到峰值时,pHAV-4AngⅡs组SHRs跟PBS组SHRs进行比较,平均收缩压降低约23mmHg,平均舒张压降低约12mmHg,血清中Angll的平均浓度降低约87pg/ml。利用Pearson双变量相关性分析对Angll浓度,血压,抗Angll抗体滴度三者进行统计学分析,结果显示：Angll浓度与血压之间存在正相关性；Angll浓度和抗Angll抗体滴度之间存在负相关性；血压和抗Angll抗体滴度存在负相关性。
本试验最终表明：以Angll为功能表位,HAV-VLP为载体的嵌合蛋白pHAV-4AngIIs具有很好的免疫原性和抗原性,可诱导机体产生较高滴度的抗AngⅡ的特异性抗体,该抗体能够有效中和血清中过多的AngⅡ,阻断RAAS,对SHRs具有一定的降压治疗效果。这为高血压疫苗的研究和开发提供了一种新的思路,具有广阔的应用前景。
Hypertension (HTN) is a chronic disease characterized by the persistent high arterial blood pressure, always accompanied the disorder of fat and carbohydrate metabolism, and accompanied the organs or tissues remodeling of heart, brain, kidneys and retina. High rates of disability and mortality are associated with HTN's complications, which include stroke, myocardial infarction, cardiac failure and chronic kidney disease. As reported, nearly one billion people currently have hypertension, which kills8million people every year worldwide. In china, there are two billion hypertensive patients at least, about10million new patients each year. Currently, hypertensive patients are treated with a blockers, calcium channel blockers (CCBs), ACE inhibitors (ACEIs) and angiotensin Ⅱ receptor antagonists. Although these medicines showed good therapeutic effect, hypertension cannot be effectively controlled due to their serious side-effects, the emergence of resistance to medicines and the poor compliance. So the cardiovascular researchers and clinical workers worldwide are actively exploring new treatments. The development of efficient, safe vaccine becomes a new research direction for the treatment and control of hypertension.
The renin-angiotensin-aldosteron system (RAAS) is the most important system involved in the onset of hypertension. AngⅡ plays an important role in this system; it causes a series of physiological effects by binding with the AngⅡ type I receptor (AT1R) on the blood vessels, heart, brain and kidneys to increase blood pressure. AngⅡ is one of the most powerful active substances to cause strong vasoconstriction and increase blood pressure. If there are anti-AngⅡ-specific antibodies in vivo, which can neutralize the serum excessive Angll and keep it on the normal level, blood pressure could be reduced. In this study, AngⅡ was selected as target to explore a novel anti-hypertension therapeutic vaccine. This vaccine is a chimeric protein which presents four successive repeated AngⅡs as the functional epitope on the surface of the hepatitis A virus-like particle (HAVLP) to elevate the immunogenicity of AngⅡ. We hope that the vaccine can activate the immune response of the host, induce the anti-AngⅡ specific neutralizing antibody to block the RAAS, and blood pressure subsequently can be decreased after immunizations.
In this study, four-artificial-repeated AngⅡ s DNA sequence was inserted into the2A region of plasmid pT7-18f obtained by modifying the HAV genome. The recombinant plasmid pT7-18f was used as template to amplify the gene P1-2A-4AngⅡs and the gene3ABC by PCR. These two gene fragments were inserted into the multiple clone sites located downstream of the PH and P10promoters, respectively, on pFastBacTM-dual vectors. The recombinant pFastBac-dual plasmids were transformed into DH10E.coli for transposition into the bacmid to obtain the recombinant bacmids. Then sf9cells were transfected with the recombinant bacmids using cellfectinⅡ reagent to obtain recombinant baculoviruses Bav-P1-2A-4AngIIs-3ABC. These recombinant plasmids above were identified by PCR, double enzyme digestion and gene sequencing methods. The recombinant baculoviruses were identified by observing cytopathic effect (CPE), PCR, gene sequencing and transmission electron microscope, and the plaque assay was used to determine the titer of recombinant baculoviruses. In this study, the target protein pHAV-4AngⅡs was expressed by Bac-to-Bac baculovirus expression system, identified through RT-PCR, SDS-PAGE, indirect immunofluorescence, western blot, transmission electron microscope and other methods, and purified by sucrose density gradient ultracentrifugation. Spontaneously hypertensive rats (SHRs) were immuned with the purified pHAV-4AngⅡs. Blood pressure in SHRs was measured by tail-cuff arterial blood pressure measurement method. The serum AngⅡ levels and the anti-AngⅡ specific antibody in sera were tested by ELISA. At last, these data were analysised though T test and Pearson bivariate correlation analysis to study immunogenicity and treatment effect of pHAV-4AngIIs.
In this study, the recombinant baculoviruses carrying P1-2A-4AngⅡs and3ABC genes were successfully constructed. It was identified using PCR, and P1-2A-4AngⅡs and3ABC gene fragments were specifically amplified. The result of gene sequencing showed that the sequences and the reading frames are correct. The typical baculovirus particles were observed under the transmission electron microscope, and the plaque assay suggested that titer of the P3viruses was approximately4×108pfu/ml. PHAV-4AngⅡs were successfully expressed in sf9cells which were infected the recombinant baculoviruses. Through RT-PCR, P1-2A-4AngIIs and3ABC genes were amplified using sf9cells infected with recombinant baculoviruses and could not be amplified using normal sf9cells. These findings suggested that these two genes can be successfully transcribed in infected sf9cells. Western blot exposed a specific protein band at a molecular mass of～37KD and the protein band may represent the VP1-2A-4AngⅡs subunit of pHAV-4AngⅡs. Specifically, fluorescence were observed in infected sf9cells, and no fluorescence were observed in normal cells, suggesting that pHAV-4AngⅡs were successfully expressed in the infected sf9cells, which provides further evidence of good immunoreactivity. The VLPs of pHAV-4AngIIs were observed and approximately22nm under transmission electron microscope. The results from ELISA and western blot suggested that the optimum expression of pHAV-4AngIIs:MOI=1, time=96h. SHRs were immunized with the purified pHAV-4AngIIs to test immunogenicity and pharmacodynamic action. The results showed that this vaccine can induce high titer anti-AngⅡ-specific IgG antibody for almost10weeks. When antibody titer reach the peak at8th week, compared the PBS group SHRs, the mean systolic blood pressure (SBP) on pHAV-4AngⅡs group SHRs degraded approximately23mmHg, the mean diastolic blood pressure (DBP) degraded approximately12mmHg, and the mean concentration of AngⅡ in sera degraded approximately87pg/ml. The results of Pearson bivariate correlation analysis showed that there is a direct correlation between AngⅡ level and blood pressure, an inverse correlation between AngⅡ level and anti-AngⅡ-specific antibody titer, an inverse correlation between blood pressure and anti-AngⅡ-specific antibody titer.
In conclusion, the chimeric protein pHAV-4AngIIs presenting AngⅡ as functional epitope on the surface of HAVLP have good immunogenicity and antigenicity, and also have good effect on reduction of blood pressure in SHRs. It can induce high titer of anti-AngⅡ-specific antibody which can neutralize·excessive AngⅡ in sera, block RAAS and lower blood pressure. These findings provide that the pHAV-4AngIIs would be a new direction of exploration for the development of anti-hypertension therapeutic vaccine.
引文
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