| 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. |
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