CN106367401A - 具有基因协同元件的病毒及其应用 - Google Patents
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Abstract
本发明公开的是具有基因协同元件的病毒及其应用。本发明包括病毒载体,所述病毒载体上插入有病毒复制子和载体特异性复制单元,所述病毒复制子包括复制子复制基因,用于启动转录翻译复制子复制基因的启动子一,连接在复制子复制基因上的基因协同元件,以及设置在病毒复制子末端的加尾信号序列;该基因协同元件由细胞因子序列、热休克蛋白序列HSPs和CpG基序中的一种以上序列构成,该基因协同元件还设置有用于启动转录翻译基因协同元件的启动子二。本发明具有能更好地主动有效激发机体特异性免疫,进而更好地发挥疾病预防或治疗效果等优点。
Description
技术领域
本发明涉及一种病毒,具体涉及的是一种具有基因协同元件的病毒及其应用。
背景技术
同类技术原理的已公开的专利分两类,一类是靶向病毒,如专利CN2004800131515和CN2014101476528中的记载,靶向病毒可特异在肿瘤细胞中复制,必须靶向肿瘤给药才能达到一定的疗效,利用病毒直接杀灭肿瘤细胞。该技术是利用腺病毒作为载体,加入一些凋亡基因可以导致肿瘤细胞死亡,该腺病毒可以特异在肿瘤细胞中复制,但这种技术的局限性在于靶向病毒容易被机体抗体识别并杀灭而失效。该技术可诱导增强机体肿瘤特异性免疫能力有限。
另一类是腺病毒甲病毒杂交载体,如专利CN2005800161337、US20070224170和WO2005112541A中的记载,腺病毒甲病毒杂交载体通过肿瘤靶向给药,在肿瘤特异性启动子启动甲病毒复制子表达,从而诱导肿瘤细胞发生凋亡。该技术利用腺病毒作为载体,加入了甲病毒复制子,促进肿瘤细胞凋亡,但该技术缺乏病毒复制元件无法在癌细胞中实现病毒复制,发挥作用效能比较慢,且疗效较低。且该类技术多次给药时同样具有第一类的局限性,即该病毒容易被机体抗体识别而杀灭而失效。该技术可诱导增强机体肿瘤特异性免疫能力同样有限。
发明内容
本发明的目的在于克服现有技术中的病毒疗效较低的问题,提供能更好地主动有效激发机体特异性免疫,进而更好地发挥疾病预防或治疗效果的具有基因协同元件的病毒,并公开了该具有基因协同元件的病毒的应用。
为达到上述目的,本发明的技术方案如下:
一种新型的杂交微生物,包括病毒载体,所述病毒载体上插入有病毒复制子和载体特异性复制单元,所述病毒复制子包括复制子复制基因,用于启动转录翻译复制子复制基因的启动子一,连接在复制子复制基因上的基因协同元件,以及设置在病毒复制子末端的加尾信号序列;
该基因协同元件由细胞因子序列、热休克蛋白序列HSPs和CpG基序中的一种以上序列构成,该基因协同元件还设置有用于启动转录翻译基因协同元件的启动子二。
通过本发明上述结构的优化,可以有效用于疾病的预防或治疗用途,且本发明的该病毒在预防和治疗上效果十分显著。发明结构的优化,能更好地主动有效激发机体特异性免疫,进而更好地发挥疾病预防或治疗效果,该疾病包括:疫病、自身代谢病、肿瘤等。
更进一步地,所述基因协同元件由细胞因子序列、热休克蛋白序列HSPs和CpG基序中的两种以上序列串联构成。
通过在本发明中插入两种以上序列的基因协同元件的作用,能使基因协同元件中的各元件达到相互促进效果,能更好地达到显著促进预防和治疗的目的。
进一步,所述复制子复制基因和基因协同元件之间连接有标签蛋白。所述病毒复制子末端的加尾信号序列位置处还连接有PolyA序列,该PolyA序列中包含连续70~200个腺苷。
其中,所述病毒载体包括但不限于以下类型中的一种,包括腺病毒,如犬腺病毒I型和II型,腺病毒2型Ad2、腺病毒5型Ad5、腺病毒35型Ad35;疱疹病毒,如人单纯疱疹病毒1型HSV1、伪狂犬病毒PRV;腺相关病毒AAV,如AAV2;痘病毒,如人痘病毒、牛痘病毒、羊痘病毒;逆转录病毒;慢病毒等。
所述启动子一为启动转录翻译病毒复制子,该启动子其来源包括但不限于以下类型中的一种,包括转录因子E2F;癌胚抗原CEA;端粒酶TERT;甲胎蛋白AFP;翻译延长因子EF-1;三磷酸甘油醛脱氢酶GAPDH;前列腺特异性抗原,如PSA;乳腺癌特异性抗原,如CA-153;人巨细胞病毒HCMV;肌动蛋白actin等。
所述复制子复制基因来源于单股正链RNA病毒(ssRNA positive-strand virus),该病毒类型包括但不限于以下类型中的一种,包括甲病毒属病毒Alphavirus,如东方马脑炎病毒Eastern equine encephalitis virus、森林脑炎病毒Semliki Forest virus、委内瑞拉马脑炎病毒Venezuelan equine encephalomyelitis virus;辛德毕斯病毒Sindbisvirus、鲑鱼胰腺病病毒Salmon pancreas disease virus、盖他病毒Getah virus、基孔肯雅病毒Chikungunya virus;风疹病毒属病毒Rubivirus,如风疹病毒Rubella virus;星状病毒Astrovirus,如哺乳动物星状病毒Mamastrovirus、禽星状病毒Avastrovirus;杯状病毒Calicivirus,如兔杯状病毒Lagovirus、诺如病毒Norovirus、人杯状病毒Sapovirus、动物杯状病毒Vesivirus;普罗维登斯病毒Providence virus;黄病毒Flavivirus,如黄热病毒Yellow fever virus、寨卡病毒Zika virus、登革热病毒Dengue virus、乙型脑炎病毒Japanese encephalitis virus、丙型肝炎病毒Hepatitis C virus、边界病病毒Borderdisease virus、牛病毒性腹泻Bovine viral diarrhea virus、猪瘟病毒Classical swinefever virus;戊型肝炎病毒Hepatitis E virus;动脉炎病毒Arterivirus,如马动脉炎病毒Equine arteritis virus、蓝耳病毒Porcine reproductive and respiratorysyndrome virus、猴出血热病毒Simian hemorrhagic fever virus;冠状病毒Coronaviridae,如传染性胃肠炎病毒Transmissible gastroenteritis virus、猪流行性腹泻病毒Porcine epidemic diarrhea virus;小核糖核酸病毒Picornaviridae,如口蹄疫病毒Foot-and-mouth disease virus、鸭肝炎病毒Duck hepatitis A virus、脑心肌炎病毒Encephalomyocarditis virus、肠病毒A型Enterovirus A、肠病毒B型Enterovirus B、肠病毒C型Enterovirus C、肠病毒D型Enterovirus D、肠病毒E型Enterovirus E、肠病毒F型Enterovirus F、肠病毒G型Enterovirus G、肠病毒H型Enterovirus H、肠病毒J型Enterovirus J、人肠病毒Human enterovirus、柯萨奇病毒Coxsackievirus、艾柯病毒Echovirus、人脊髓灰质炎病毒Human poliovirus、鼻病毒A型Rhinovirus A、鼻病毒B型Rhinovirus B、鼻病毒C型Rhinovirus C、马B型鼻病毒Equine rhinitis B virus、人甲肝病毒Hepatitis A virus、捷申病毒Porcine teschovirus、禽脑脊髓炎病毒Avianencephalomyelitis virus等。
该复制子复制基因为原始病毒复制子上的复制酶蛋白序列,如甲病毒SFV的NSP-1~NSP-4。
所述标签蛋白为绿色荧光蛋白,可替换为半乳糖甘酶LacZ,荧光素酶Luciferase,肿瘤相关抗原,致病原相关的抗原等。肿瘤相关抗原如:EGFR、VEGFR、PDGFR、AFP、PSA等。致病原相关的抗原如:细菌或病毒等微生物来源的蛋白或核酸、人或动物自身来源的表达异常的蛋白或核酸。
所述启动子二用于启动转录翻译基因协同元件,该启动子其来源包括但不限于以下类型中的一种,包括翻译延长因子EF-1;转录因子E2F;癌胚抗原CEA;端粒酶TERT;甲胎蛋白AFP;前列腺特异性抗原,如PSA;乳腺癌特异性抗原,如CA-153;人巨细胞病毒HCMV;肌动蛋白actin;三磷酸甘油醛脱氢酶GAPDH等。
基因协同元件:三种基因中的任意两种以及两种以上的基因串联后协同作用,两种以及两种以上的基因之间相互促进,达到相互促进的协同效果。其中,三种基因包括细胞因子序列、热休克蛋白序列HSPs和CpG基序。
其中,细胞因子序列来源包括但不限于以下类型中的一种,包括IL-2,IL-3,IL-12,IL-15,IL-23,IL-27,IL-30,IL-35,G-CSF,M-CSF,GM-CSF,IFN等。
热休克蛋白序列HSPs包括HSP110,HSP90,HSP70,HSP65,HSP60以及小分子热休克蛋白Small heat shock proteins(sHSPs)。热休克蛋白序列HSPs来源包括但不限于以下类型中的一种,包括分支杆菌科Mycobacteriaceae,如牛结核分支杆菌Mycobacteriumbovis、人结核分支杆菌Mycobacterium tuberculosis、鸟分支杆菌Mycobacterium avium等;棒状杆菌科Corynebacteriaceae;迪茨氏菌科Dietziaceae;戈登氏菌科Gordoniaceae;诺卡氏菌科Nocardiaceae;束村氏菌科Tsukamurellaceae;慢反应脂肪酸菌科Segniliparaceae;梭菌科Clostridiaceae,如肉毒杆菌Clostridium botulinum等。
CpG基序的来源包括但不限于以下类型中一种,包括人、大鼠、小鼠、猪、鸡、牛、羊、犬、猫等。
病毒复制子末端的加尾信号序列为bGH polyA signal。PolyA为连续70~200个腺苷,该PolyA序列对转录mRNA起到稳定的作用。
进一步,所述病毒复制子与病毒载体之间还插入有载体特异性复制单元,所述载体特异性复制单元包括载体复制基因,用于启动转录翻译载体特异性复制单元的启动子三,以及设置在载体特异性复制单元末端的加尾信号序列。
载体特异性复制单元:该元件含启动子三,载体复制基因和加尾信号序列三部分组成。该元件为条件性必需元件,仅在肿瘤治疗领域用途中使用,除肿瘤治疗领域外其他用途不需要该元件。
其中,启动子三用于启动转录翻译载体特异性复制单元,该启动子其来源包括但不限于以下类型中的一种,包括癌胚抗原CEA;转录因子E2F;端粒酶TERT;甲胎蛋白AFP;前列腺特异性抗原,如PSA;乳腺癌特异性抗原,如CA153等。
载体复制基因:病毒载体复制必需的基因,如腺病毒的E1A基因等。
载体特异性复制单元末端的加尾信号序列为SV40polyA signal。
本发明通过增加载体特异性复制单元,在原有主动有效激发机体特异性免疫效应基础上,增加该复制单元所具备的主要功能,即靶向病毒作用,在肿瘤细胞中复制增值从而直接靶向杀灭肿瘤细胞。本发明即同时具备第一类的靶向病毒和第二类腺病毒甲病毒杂交载体的优点,并达到克服其各自缺点的效果,该疗效比第一类、第二类优势更明显,效果十分显著。
本发明还提供了如上述具有基因协同元件的病毒在主动有效增强机体特异性免疫和靶细胞杀灭中的应用,该靶细胞包括疫病病原感染细胞、自身代谢病相关细胞、肿瘤细胞等。
即,不具有载体特异性复制单元的具有基因协同元件的病毒,其能够主动有效增强机体特异性免疫,进而在疾病预防或治疗用途中应用,该疾病包括疫病、自身代谢病、肿瘤等。
而具有载体特异性复制单元的具有基因协同元件的病毒,其能够有效主动增强机体特异性免疫和靶向杀灭癌细胞双重作用,进而在肿瘤治疗用途中应用。肿瘤治疗用途中,具有载体特异性复制单元的具有基因协同元件的病毒的带来的抗肿瘤效果比不具有载体特异性复制单元的具有基因协同元件的病毒更好。
本发明与现有技术相比,具有以下优点及有益效果:
1、本发明的病毒能更好地有效主动激发机体特异性免疫,进而更好地发挥疾病预防或治疗效果;同时,本发明的病毒还能具备第一类的靶向病毒和第二类腺病毒甲病毒杂交载体的优点,并同时达到克服其各自缺点的效果,该疗效比第一类、第二类优势更明显,效果十分显著;
2、本发明基因协同元件通过细胞因子序列、热休克蛋白序列HSPs和CpG基序三种基因中的任意两种以及两种以上的基因串联后协同作用,两种以及两种以上的基因之间相互作用,达到相互促进的协同效果;
3、本发明在肿瘤领域中,不仅可以靶向直接杀灭肿瘤细胞,与此同时,由于加入了基因协同元件,从而协同主动有效增强机体特异性免疫,利用机体特异性免疫高效杀灭肿瘤细胞。
附图说明
图1为本发明病毒的结构示意图。
图2为实施例2采用CCK-8试剂检测小鼠淋巴细胞增殖的结果示意图。
图3为实施例4采用CCK-8试剂检测小鼠淋巴细胞增殖的结果示意图。
具体实施方式
下面结合实施例,对本发明作进一步地详细说明,但本发明的实施方式不限于此。
实施例1
具有基因协同元件的病毒,由顺次串联的启动子一、复制子复制基因、标签蛋白、启动子二、细胞因子序列、热休克蛋白序列HSPs、CpG基序、PolyA序列、加尾信号序列、启动子三、载体复制基因、加尾信号序列,共同插入到病毒载体上构成,全称为Ad-SFV-ABC-E1A,其结构如图1所示。
其中,病毒载体采用腺病毒的pShuttle-CMV载体,并采用BglII-HindIII进行酶切处理。酶切后插入位点两端位置处的基因序列如下:
5'-...CATCATCAATATTATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTTATGGAGTTTGTCAC-3';
5'-AACGCGGATCTGGGCGTGGTTAAGGGTGGGAAAGAATATATAAGGT GGGGGTCTTATG...-3'。
本实施例中启动子一采用mEF1启动子-BglII,复制子复制基因由甲病毒复制子采用KpnI-XhoI酶切后获得的pSFV基因序列构成,标签蛋白采用GFP标签-ApaI,启动子二为mE2F启动子,细胞因子序列采用mGM-CSF,热休克蛋白序列HSPs采用牛结核分枝杆菌HSP70,CpG基序采用CpG岛,PolyA序列采用连续70个腺苷,PolyA序列后面的加尾信号序列采用bGHpolyA signal,启动子三采用pCEA启动子,载体复制基因为E1A序列,载体复制基因后面的加尾信号序列采用SV40polyA signal。本实施例中病毒的具体基因序列如SEQ ID NO:1所示。
本实施例的具体构建方法如下:
提取小鼠基因组DNA,设计mEF1启动子特异性引物,上下游引物分别含BglII与KpnI酶切位点,经PCR产物测序验证后得到913bp mEF1启动子,该mEF1启动子如SEQ ID NO:8所示,将mEF1启动子插入到含BglII、KpnI酶切线性化的PUC57质粒中得到3553bp pmEF1。该pUC57质粒中含BglII、KpnI、XhoI、HindIII等酶切位点。
pSFV质粒改造后使其sfv复制子上下游分别含KpnI和XhoI酶切位点,其经SspI酶切后线性化。以pEGFP-N1质粒为模板,设计GFP特异性引物,经PCR产物测序验证后得到720bp GFP,该GFP的序列结构如SEQ ID NO:7所示。设计mE2F启动子特异性引物,经PCR产物测序验证后得到193bp mE2F启动子,该mE2F启动子如SEQ ID NO:9所示。GFP与mE2F经融合PCR得到913bp的GFP-mE2F产物。
从细胞系RAW 264.7中提取RNA为模板,设计mGM-CSF特异性引物,经PCR产物测序验证后得到423bp的mGM-CSF基因(细胞因子序列),mGM-CSF基因如SEQ ID NO:4所示。以结核菌为样本提取细菌基因组DNA,设计HSP70特异性引物,经PCR产物测序验证后得到1482bpHSP70基因(HSPs);基因合成438bp的CpG岛-polyA-bGH polyA signal,该CpG岛序列如SEQID NO:2所示。
mGM-CSF、HSP70与CpG岛-polyA-bGH polyA signal融合PCR后得到2343bp的ABC产物,再由GFP-mE2F与ABC经融合PCR后得到3256bp的GFP-mE2F-ABC产物。该GFP-mE2F-ABC产物与SspI线性化的pSFV进行同源重组得到pSFV-GFP-mE2F-ABC,该pSFV的序列如SEQ IDNO:10所示,该质粒经KpnI和XhoI双酶切后的11174bp基因片段,插入到同样双酶切的线性化pmEF1中得到14715bp的pmEF1-SFV-ABC,pmEF1-SFV-ABC由XhoI、HindIII酶切线性化。
小鼠基因组DNA为模板扩增出的297bp的pCEA启动子、293T细胞提取的DNA为模板扩增出的850bp的E1A基因以及122bp的SV40polyA signal,经融合PCR得到产物为1289bp的CEA-E1A基因片段,与XhoI、HindIII酶切后插入线性化的pmEF1-SFV-ABC载体中,连接后得到15992bp的pmEF1-SFV-ABC-E1A。
该质粒pmEF1-SFV-ABC-E1A由BglII、HindIII酶切后得到13400bp mEF1-SFV-ABC-E1A基因片段,与该双酶切的改造pShuttle-CMV(不含cmv增强子和启动子)线性化后连接,得到20029bp pShuttle-mEF1-SFV-ABC-E1A质粒。
由PmeI酶切该pShuttle-mEF1-SFV-ABC-E1A质粒得到线性化质粒,与pAdeasy-1质粒共转BJ5183菌进行菌内同源重组,筛选阳性质粒49.5kb pAd-SFV-ABC-E1A。该质粒由PacI酶切线性化后,得到46.5kb线性化质粒转染293T细胞,经细胞培养、病毒鉴定与扩增纯化后得到杂合腺病毒Ad-SFV-ABC-E1A,该Ad-SFV-ABC-E1A的序列如SEQ ID NO:1所示。
实施例2
本实施例与实施例1的区别在于:本实施例在实施例1所述的Ad-SFV-ABC-E1A上去掉部分基因序列,具体设置如下:
肿瘤药物1组:Ad-SFV-ABC-E1A。
肿瘤药物2组:在Ad-SFV-ABC-E1A基础上去掉CpG基序,该CpG基序为CpG岛,该CpG岛序列如SEQ ID NO:2所示;
肿瘤药物3组:在Ad-SFV-ABC-E1A基础上去掉热休克蛋白序列HSPs,该热休克蛋白序列HSPs为牛结核分枝杆菌HSP70,该牛结核分枝杆菌HSP70的序列如SEQ ID NO:3所示;
肿瘤药物4组:在Ad-SFV-ABC-E1A基础上去掉细胞因子序列,该细胞因子序列为mGM-CSF,该mGM-CSF的序列如SEQ ID NO:4所示;
肿瘤药物5组:在Ad-SFV-ABC-E1A基础上去掉热休克蛋白序列HSPs和CpG基序;
肿瘤药物6组:在Ad-SFV-ABC-E1A基础上去掉细胞因子序列、CpG基序;
肿瘤药物7组:在Ad-SFV-ABC-E1A基础上去掉细胞因子序列、热休克蛋白序列HSPs;
肿瘤药物8组:在Ad-SFV-ABC-E1A基础上去掉细胞因子序列、热休克蛋白序列HSPs和CpG基序。
本发明通过下述试验方式验证本发明的结果。
1、病毒在BALB/C鼠体内治疗肿瘤移植瘤
将4-5周龄的雌性BALB/C鼠皮下途径接种肺癌细胞LLC,每只鼠为1×106细胞,1-2周后当肿瘤达到100~150mm3进行动物分为九组。每组分别给予1×109pfu/mice的上述八种肿瘤药物进行治疗,剩余一组为PBS组,进行尾静脉注射,对肿瘤体积(mm3)进行检测,检测结果如表1所示。
表1
1组 | 2组 | 3组 | 4组 | 5组 | 6组 | 7组 | 8组 | PBS组 | |
1周 | 105 | 84 | 75 | 88 | 96 | 106 | 98 | 97 | 101 |
2周 | 136 | 175 | 173 | 170 | 152 | 152 | 145 | 180 | 174 |
3周 | 195 | 182 | 205 | 200 | 188 | 195 | 203 | 215 | 235 |
4周 | 198 | 167 | 170 | 167 | 189 | 204 | 251 | 276 | 318 |
5周 | 185 | 205 | 193 | 172 | 161 | 218 | 249 | 314 | 380 |
6周 | 111 | 193 | 162 | 120 | 172 | 208 | 211 | 355 | 504 |
7周 | 79 | 122 | 152 | 107 | 124 | 218 | 197 | 465 | 812 |
8周 | 16 | 91 | 66 | 87 | 107 | 107 | 162 | 425 | 997 |
9周 | 0 | 45 | 25 | 31 | 64 | 56 | 117 | 453 | 1355 |
通过1的试验结果所示,肿瘤药物1组至肿瘤药物7组均能够更早地抑制肿瘤的生长。肿瘤药物1组组最优,8周后肿瘤基本消失;肿瘤药物2组至肿瘤药物7组肿瘤体积大幅减小;肿瘤药物8组在7至9周肿瘤大小基本维持一定水平;PBS组肿瘤持续生长导致体积越来越大。
2、CCK-8试剂检测小鼠淋巴细胞增殖
将6-8周龄的BALB/C小鼠,随机分为9组,1-8组分别对应注射1×109pfu/mice的上述八种肿瘤药物,剩余1组为PBS组,进行尾静脉注射。14d、28d分别按常规方法分离小鼠PBMC,将新鲜分离的PBMC计数后,将细胞用RPMI-1640完全培养液调整细胞浓度至107个/mL。将上述制备的淋巴细胞悬液混匀加入96孔细胞培养板,每孔加入淋巴细胞悬液100μL。将每组淋巴细胞悬液设3个重复,分别用肺癌细胞冻融物去刺激。将细胞置于37℃,5%CO2培养箱培养72h后,每孔加入10μL CCK-8试剂,混匀后继续培养4h,测定OD450nm值,计算刺激指数(SI)=OD(刺激孔)/OD(空白对照孔)。检测结果如图3所示。
通过图2可知,致病原药物1组至致病原药物7组14d时刺激指数超过2,以病原药物1组效果最优;对照组致病原药物8组28d时刺激指数在2以内;PBS组没有产生特异性淋巴细胞增殖。
并且,通过表1和图2的结果可知,当基因协同元件中采用两种以上序列串联构成时,能达到相互促进的效果,使特异性淋巴细胞增殖的预防效果以及肿瘤的治疗效果更加显著。
实施例3
本实施例与实施例1的区别在于,本实施例的病毒中不包括载体特异性复制单元,并将GFP序列替换为小鼠细小病毒MVM-VP2,本实施例中该MVM-VP2的具体序列结构如SEQID NO:5所示,该载体特异性复制单元的基因序列如SEQ ID NO:6所示。
实施例4
本实施例与实施例1的区别在于,本实施例在实施例1所述的Ad-SFV-ABC上去掉部分基因序列,具体设置如下:
致病原药物1组:Ad-SFV-ABC。
致病原药物2组:在Ad-SFV-ABC基础上去掉CpG基序,该CpG基序为CpG岛,其CpG岛序列如SEQ ID NO:2所示;
致病原药物3组:在Ad-SFV-ABC基础上去掉热休克蛋白序列HSPs,该热休克蛋白序列HSPs为牛结核分枝杆菌HSP70,该牛结核分枝杆菌HSP70的序列如SEQ ID NO:3所示;
致病原药物4组:在Ad-SFV-ABC基础上去掉细胞因子序列,该细胞因子序列为mGM-CSF,该mGM-CSF的序列如SEQ ID NO:4所示;
致病原药物5组:在Ad-SFV-ABC基础上去掉热休克蛋白序列HSPs和CpG基序;
致病原药物6组:在Ad-SFV-ABC基础上去掉细胞因子序列、CpG基序;
致病原药物7组:在Ad-SFV-ABC基础上去掉细胞因子序列、热休克蛋白序列HSPs;
致病原药物8组:在Ad-SFV-ABC基础上去掉细胞因子序列、热休克蛋白序列HSPs和CpG基序。
本发明下述试验方式验证本发明的结果。
1、小鼠细小病毒MVM-VP2特异性抗体检测
将6-8周龄的小鼠,随机分为9组,1-8组腿部肌肉途径对应的分别注射1×109pfu/mice上述八组致病原药物进行免疫,剩余一组为PBS组,进行尾静脉注射,免疫前、免疫后每周,采血分离血清,用制备的小鼠细小病毒MVM-VP2抗体间接ELISA检测,检测MVM-VP2特异性中和抗体产生情况。抗体滴度(%)的检测结果如表2所示。
表2
1组 | 2组 | 3组 | 4组 | 5组 | 6组 | 7组 | 8组 | PBS组 | |
3d | 3.5 | 2.2 | 1.7 | 2.8 | 1.8 | 1.9 | 1.5 | 1.1 | 0.5 |
7d | 15.8 | 11.8 | 10.7 | 12.2 | 8.3 | 9.2 | 10.4 | 5.3 | 0.5 |
14d | 21.2 | 17.9 | 19.8 | 20.3 | 13.1 | 14.4 | 18.7 | 8.8 | 0.5 |
21d | 36.4 | 35.0 | 30.3 | 26.2 | 25.9 | 24.0 | 24.6 | 16.5 | 0.6 |
28d | 51.7 | 52.3 | 44.8 | 46.7 | 39.0 | 41.2 | 42.6 | 28.0 | 0.5 |
35d | 64.4 | 58.5 | 60.6 | 60.9 | 52.6 | 57.5 | 55.5 | 38.7 | 0.8 |
42d | 72.8 | 65.1 | 66.8 | 68.1 | 60.4 | 59.7 | 61.7 | 45.0 | 0.9 |
通过表2可知,3d-7d之间1组的抗体滴度起峰最快,在整个42d监测过程中各监测点滴度值最高;2组至7组的整体表现较好;8组起峰较慢、抗体滴度值较小;PBS组检测不到特异性抗体。
2、CCK-8试剂检测小鼠淋巴细胞增殖
本检测方法中采用原核表达MVM-VP2蛋白作为刺激原。
将6-8周龄的balb/c小鼠,随机分为9组,1-7组分别对应的注射1×109pfu/mice的上述八组致病原药物,剩余一组为PBS组,进行尾静脉注射。14d、28d分别按常规方法分离小鼠PBMC,将新鲜分离的PBMC计数后,将细胞用RPMI-1640完全培养液调整细胞浓度至107个/mL。将上述制备的淋巴细胞悬液混匀加入96孔细胞培养板,每孔加入淋巴细胞悬液100μL。将每组淋巴细胞悬液设3个重复,分别用原核表达MVM-VP2蛋白去刺激。将细胞置于37℃,5%CO2培养箱培养72h后,每孔加入10μL CCK-8试剂,混匀后继续培养4h,测定OD450nm值,计算刺激指数(SI)=OD(刺激孔)/OD(空白对照孔)。
上述检测方法的检测结果如图3所示,通过图3可知:1组效果最优;药物组1-7组表现较好;8组刺激指数较差;PBS组没有产生特异性淋巴细胞增殖。
并且,通过通过表2和图3的结果可知,当基因协同元件中采用两种以上序列串联构成时,能达到相互促进的效果,使特异性淋巴细胞增殖的预防效果更好,特异性抗体的抗体滴度更高。
上述实施例仅为本发明的优选实施例,并非对本发明保护范围的限制,但凡采用本发明的设计原理,以及在此基础上进行非创造性劳动而作出的变化,均应属于本发明的保护范围之内。
Claims (10)
1.具有基因协同元件的病毒,包括病毒载体,其特征在于:
所述病毒载体上插入有病毒复制子,所述病毒复制子包括复制子复制基因,用于启动转录翻译复制子复制基因的启动子一,连接在复制子复制基因上的基因协同元件,以及加在病毒复制子末端的加尾信号序列;
该基因协同元件由细胞因子序列、热休克蛋白序列HSPs和CpG基序中的一种以上序列构成,该基因协同元件还设置有用于启动转录翻译基因协同元件的启动子二。
2.根据权利要求1所述的具有基因协同元件的病毒,其特征在于,该基因协同元件由细胞因子序列、热休克蛋白序列HSPs和CpG基序中的两种以上序列串联构成。
3.根据权利要求1所述的具有基因协同元件的病毒,其特征在于,
所述复制子复制基因和基因协同元件之间连接有标签蛋白。
4.根据权利要求1所述的具有基因协同元件的病毒,其特征在于,
所述细胞因子序列选自IL-2,IL-3,IL-12,IL-15,IL-23,IL-27,IL-30,IL-35,G-CSF,M-CSF,GM-CSF,IFN中的一种;
所述热休克蛋白序列HSPs选自HSP110,HSP90,HSP70,HSP65,HSP60以及sHSPs中的一种。
5.根据权利要求1所述的具有基因协同元件的病毒,其特征在于,
所述病毒载体选自腺病毒、疱疹病毒、腺相关病毒、痘病毒、逆转录病毒和慢病毒中的一种;
所述复制子复制基因的来源为单股正链RNA病毒的基因序列;
所述病毒复制子末端的加尾信号序列为bGH polyA加尾信号。
6.根据权利要求1所述的具有基因协同元件的病毒,其特征在于,所述病毒复制子末端的加尾信号序列位置处还连接有PolyA序列,该PolyA序列中包含连续70~200个腺苷。
7.根据权利要求1~6任一项所述的具有基因协同元件的病毒,其特征在于,
所述病毒复制子与病毒载体之间还插入有载体特异性复制单元,所述载体特异性复制单元包括载体复制基因,用于启动转录翻译载体特异性复制单元的启动子三,以及设置在载体特异性复制单元末端的加尾信号序列。
8.根据权利要求7所述的具有基因协同元件的病毒,其特征在于,所述载体特异性复制单元末端的加尾信号序列为SV40polyA加尾信号。
9.如权利要求1~6任一项所述具有基因协同元件的病毒主动有效增强机体特异性免疫在疾病预防或治疗用途中的应用。
10.如权利要求7~8任一项所述具有基因协同元件的病毒主动有效增强机体特异性免疫和靶向杀灭癌细胞在肿瘤治疗用途中的应用。
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