CN103992396A - 一种潜在的高效重组HIV-1 CRF07-BC gp140免疫原的制备方法 - Google Patents
一种潜在的高效重组HIV-1 CRF07-BC gp140免疫原的制备方法 Download PDFInfo
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Abstract
本发明公开了一种潜在的高效重组HIV-1 CRF07-BC gp140免疫原的制备方法。该免疫原基于国际上已经发表和本实验室获得的HIV-1包膜蛋白晶体结构而设计,具体方法为运用重叠延伸PCR技术,获得gp140的基因片段,将目的基因克隆入真核表达载体pMT,经质粒大提去除内毒素后,与抗性筛选质粒pCoBlast一起共转染黑腹果蝇Schneider2(S2)细胞,用杀稻瘟菌素(Blasticidin S)进行阳性克隆筛选,筛选出稳定高效分泌表达gp140的S2细胞系。扩大培养后,经镍柱亲和层析和凝胶过滤层析两步纯化,即可获得纯度很高的gp140。经一系列生物化学和生物物理技术证明该gp140聚合状态均一,抗原反应性高,非常适合作为免疫原用于艾滋病亚单位疫苗或多价联合疫苗的研发。
Description
所属技术领域:
本发明属于生物工程领域,具体涉及到基于蛋白质结构的免疫原设计,分子克隆,脂质体转染,蛋白质表达纯化,分析型超速离心(SV-AUC)和酶联免疫吸附测定(ELISA)等技术。
背景技术:
艾滋病(accquired immunodeficiency syndrome,AIDS)是由人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染所导致的一种病死率极高的正在全球肆虐的慢性传染病。HIV分为两种:HIV-1和HIV-2,HIV-1的毒性与传染性均高于HIV-2,在全球范围内引发AIDS的流行,HIV-2则基本只存在于西部非洲部分地区。世界卫生组织网站的数据显示,截止2013年10月,全球艾滋病病毒感染者人数为3600万,累计死亡超3600万,2012年新增艾滋病病毒感染者230万,160万人因艾滋病而死亡。到2012年我国实际HIV感染人数已超过100万,中国成人流行率为0.1%~0.5%,防控形势比较严峻。
艾滋病的流行已成为严重影响人类健康和全球社会经济发展的公共卫生问题。目前仍没有针对HIV的特效治疗方法,虽然高效抗逆转录病毒的治疗方法(highly active antiretroviral therapy,HAART)已经在减轻患者痛苦、延长患者寿命等方面取得了一定的效果,但用于治疗HIV感染的药物只能控制病毒复制,不能彻底清除病毒,而且抗HIV药物价格昂贵,具有较严重的副作用,药物使用不当,也会诱发耐药株的产生。因此,研制安全、有效的疫苗是控制HIV传播的重要手段之一,被认为是预防艾滋病的最有效工具。
流行病学调查显示,HIV-1不同亚型或流行重组型在不同地域的感染者中所占比例有很大差异。而不同型别间免疫原性和抗原性的差异也导致针对某一亚型病毒的疫苗对另一亚型的病毒并无很好的交叉保护效果。这一情况给HIV疫苗的研发带来很大挑战。在我国,HIV-1最主要的流行亚型或流行重组型是CRF07-BC,CRF08-BC,B’(泰国B)和CRF01-AE,其中B’/C重组毒株(CRF07-BC、CRF08-BC)感染比例在全部感染者中逐年上升,所占比已超过50%,表明该重组病毒可能获得了某种传播优势而有加速流行的趋势,并在传播过程中逐渐取代了亲代B’和C亚型HIV-1毒株。无论从B’/C重组株在我国感染者中所占的比例还是该毒株表现出的传播优势来看,选择B’/C重组毒株主要抗原作为疫苗的免疫原都有利于迅速遏制我国艾滋病的蔓延。
有效的疫苗通常需要高效的抗体反应来阻断感染和清除抗原,因此广谱高效中和抗体的 产生对艾滋病疫苗的成功至关重要。HIV-1包膜糖蛋白(Env)的前体蛋白gp160在靶细胞的粗面内质网中起始合成、折叠并伴随着寡聚化和部分糖基化,随后被输送到高尔基体中,经furin蛋白酶家族剪切形成成熟的表面糖蛋白gp120(SU)和跨膜蛋白gp41(TM),酶切后gp120和gp41仍然非共价地联系在一起形成异二聚体并进一步糖基化,最终定位于病毒表面,形成由三个gp120分子和三个gp41分子构成的三聚体(trimer)刺突(spike)。因为包膜糖蛋白(Env)是病毒唯一暴露于外部环境中的结构,所以是人体中和抗体识别并中和的最主要的病毒成分,这样就使得Env一直以来都是HIV疫苗研发的焦点。如何进一步提高Env的免疫原性就成了摆在广大研究者面前的一个至关重要的难题。起初,大家的兴趣主要集中在单体Env组分——gp120和gp41上面,结果却很不理想。随后,大家的目光就逐渐转到了类似于天然构象的可溶的三聚体Env胞外结构域gp140上面。研究发现三聚体的gp140确实可以比单体的gp120触发更强的中和抗体反应,并且具有几乎和完整病毒刺突一样的全部抗原性质,所以三聚体gp140就成了以诱导中和抗体为主要目标的艾滋病疫苗的一个重要发展方向。目前效果最好的是BG505SOSIP.664gp140三聚体,它在野生型序列的基础上做了许多改变,包括1)A501C和T605C(参照HIV-1HXB2标准株gp160编号,下同)替换以在gp120和gp41胞外域形成二硫键,2)I559P替换以增强三聚体的稳定性,3)膜近外侧区(MPER)的删除以增强三聚体的可溶性和减少高聚体的形成,4)T332N的替换以产生依赖于该糖苷的多个广谱中和抗体的表位利于纯化,furin的自然位点(REKE)替换为最适位点(RRRRRR,R6)以利于切割完全。但是该三聚体有一个缺点,二硫键的引入虽然在稳定gp120和gp41胞外域相互作用的过程中起了十分重要的作用,但也有可能将此gp140锁定在融合前的构象(该gp140的晶体和电镜的结构支持此观点),作为免疫原并不一定利于广谱中和抗体的产生。
我们在最常规的突变掉furin的酶切位点(K/R-X-K/R-R,REKR)以获得未切割的gp140(uncleaved gp140,gp140unc)的基础上,通过基于结构的优化设计,用柔性linker将向FPPR延长多个氨基酸的gp41胞外核心结构域与两端做了截短的gp120共价连接,获得了高表达的抗原反应性(antigenicity)有较大提高的,生物化学和生物物理性质可以媲美BG505SOSIP.664gp140的中国流行株HIV-1CRF07-BC gp140,为艾滋病病毒疫苗特别是中国优势流行病毒疫苗的研制奠定了良好的基础。
发明内容:
本发明公开了一种潜在的高效重组HIV-1CRF07-BC gp140免疫原的制备方法,其特征在于我们参照本实验室发表的HIV-1CRF07gp41胞外核心结构域的晶体结构(PDB号:3WFV)和马传染性贫血病毒(EIAV)跨膜糖蛋白gp45胞外核心结构域的晶体结构(PDB号:3WP2 和3WMI)以及国际同行解析的HIV-1gp120的核心结构域的晶体结构和HIV-1Env胞外结构域gp140的晶体结构(PDB号:3TGS、3TIH和4NCO),通过向近融合肽区(fusion peptide proximal region,FPPR)延长gp41胞外核心结构域氨基末端七肽重复NHR(HR1)的多个氨基酸,再通过柔性linker与gp120共价连接,获得了gp41的六股螺旋束(six-helix bundle)闭合程度增加,稳定性提高的三聚体gp140。具体为用GGSGG将gp41的氨基末端七肽重复NHR(HR1)和羧基末端七肽重复CHR(HR2)连接起来,运用重叠延伸聚合酶链式反应(overlap extension PCR)技术获得了gp41胞外核心区的DNA片段,然后用GSGAG将gp41和gp120共价连接起来,运用overlap extension PCR技术获得了包膜糖蛋白Env胞外结构域的基因片段gp140,将目的基因克隆入真核表达载体pMT,经质粒大提去除内毒素后,与抗性筛选质粒pCoB1ast一起共转染黑腹果蝇Schneider2(S2)细胞,用杀稻瘟菌素(B1asticidin S)进行阳性筛选,筛选出稳定高效分泌表达gp140的S2细胞系扩大培养后,用镍柱结合缓冲液(50mM/LTris-HCl,500mM/LNaCl,pH8.0)替换SFX-Insect昆虫培养基,经镍柱亲和层析和凝胶过滤层析(Ge1filtration chromatography,GF)纯化,即可获得目的蛋白gp140。经聚丙烯酰胺凝胶电泳(SDS-PAGE)检测并结合凝胶过滤色谱的峰图初步判定纯化得到的三体gp140的纯度和均一性都非常好,且表达量较大。进一步利用分析型超速离心沉降速率法(SV-AUC)测定该gp140的绝对分子量以获得蛋白在溶液中的聚集状态信息,结果发现溶液中的gp140绝大部分都以三聚体形式存在,其它聚合形式包括单体的含量较少,大大优于gp140unc。最后利用酶联免疫吸附实验(ELISA)检测gp140的抗原反应性(antigenicity),发现该gp140三体的抗原反应性至少比gp140unc三体高四倍。
本发明与gp140unc三体相比,均一性好,抗原反应性(antigenicity)高,生化和物理性质可以媲美BG505SOSIP.664gp140,且针对目前中国流行株,非常适合作为免疫原用于艾滋病亚单位疫苗或多价联合疫苗的研发。
附图说明
图1是gp140构建的原理图。
图2是BG505SOSIP.664gp140的凝胶过滤图谱(引用自Rogier W.Sanders等人,2013PLoS Pathog:e1003618)。图2A是BG505SOSIP.664gp140经Hiload26/60Superdex200分了筛层析柱纯化的图谱,图2B是经BG505SOSIP.664gp140经Hiload26/60Superdex200分子筛层析柱纯化后再重过Superose610/30column分子筛层析柱的分析图谱。
图3是目的蛋白的凝胶过滤图谱与鉴定。图3A是重组蛋gp140经Hiload16/60Superdex200分子筛层析柱纯化的图谱,图3B是经Hiload16/60Superdex200分子筛层析柱纯化纯化后重 组蛋白gp140的SDS-PAGE检测,图3C是gp140蛋白经Hiload16/60Superdex200分子筛层析柱纯化后再重过Superdex20010/300分子筛层析柱的分析图谱。
图4是纯化后的gp140蛋白的Western blot检测结果。
图5是纯化的中国仓鼠卵巢细胞(CHO)表达的gp140unc的蓝绿非变性聚丙烯酰氨凝胶电泳(BN-PAGE)检测(引用自Norbert Schülke等人,2002J Virol76:7760-7776)。
图6是纯化的gp140的分析型超速离心沉降速率法分析。
图7是纯化的重组蛋白gp140与多位艾滋病长期不进展者雪清的酶联免疫吸附试验。
下面结合附图和实施例对本发明作进一步的详细叙述。
具体实施方式:
实施例1.重组gp140真核表达载体的构建
以CRF07-BC gp160cDNA为模板,经两次overlap extension PCR,获得了用两个不同linker连接的包膜糖蛋白Env胞外结构域(见图1)的DNA片段gp140,通过双酶切连接的方法将目的基因片段克隆入经本实验室改造过的pMT/Bip/TEV-HisA载体。通过将V5表位替换为烟草蚀纹病毒蛋白酶(TEV酶)的酶切位点,使我们能够用TEV酶除去目的蛋白羧基末端的组氨酸标签(6×His-tag),使目的蛋白羧基末端尽可能少地带有不必要的寡肽。经过双酶切和测序鉴定正确后(氨基酸和核苷酸序列见序列表),通过无内毒素质粒大提试剂盒进行目的质粒的大提以提高质粒浓度和去除内毒素。
载体构建过程中用到的引物为,
引物1:5’CATGCCATGG GTGTGGAAGGGCGCCACC3’
引物2:5’TCCTGCCCCTGAGCCGGGCTTGATCTCCACCAC3’
引物3:5’GGCTCAGGGGCAGGAAGCATCACCCTGACCGTGCAG3’
引物4:5’ACCGCCTGACCCTCCCTGCTGGTCCTTCAGG3’
引物5:5’GGAGGGTCAGGCGGTTGGGACAACATGACCTGG3’
引物6:5’CTAGTCTAGAGGCCAGCAGGTCCTTCTC3’
实施例2.S2细胞的共转染及稳定表达目的蛋白的S2细胞系的筛选
提前一天将细胞铺于T25培养瓶中,当细胞的汇合度(confluency)达到60-70%时用脂质体法进行共转染。转染步骤按照转染试剂Cellfectin II的说明书进行,注意重组质粒和抗性筛选质粒pCoBlast的质量比为19∶1。细胞于27℃孵育5小时后吸出转染液,加入4mL新鲜的SFX-Insect培养基,继续孵育48h,弃去原培养基,加入含有杀稻瘟菌素(终浓度为25ug/mL)的SFX-Insect培养基进行抗性筛选,未成功转染的细胞在加入杀稻瘟菌素筛选后的一周内会大量死亡,少量的贴壁细胞会在培养瓶中继续生长,大约3周左右,当细胞的汇合度达到100% 时,即可获得稳定高效分泌表达重组gp140的S2细胞系。
实施例3.重组gp140的表达纯化
将筛选出的T25S2细胞系扩大培养至T75培养瓶中(体积比1∶5),进一步转到500mL转瓶中进行扩大培养(体积比1∶10),27℃,120rpm,培养2-3天,当细胞的生长达到对数期时,加入硫酸铜诱导蛋白表达(终浓度为0.5mmol/L),3天后收集。将S2细胞收集到300mL的离心桶中,4000rpm,4℃离心15min。收集细胞上清,经0.22μm的滤膜过滤后,放至Amicon Stirred Cell8003型超滤杯中浓缩蛋白,压缩50mL时,用镍柱结合缓冲液(50mM/L Tris-HCl,500mM/L NaCl,pH8.0)稀释10倍后,将蛋白液收集至50mL离心管中,20000rpm,4℃离心20min,收集上清,经0.22μm的滤膜过滤后,将上清液进行镍柱亲和层析纯化,用镍柱洗涤缓冲液(50mM/L Tris-HCl,500mmol/L NaCl,20mM咪唑,pH8.0)冲洗三个柱长,洗去非特异性吸附的杂蛋白,最后用镍柱洗脱缓冲液(50mM Tris-HCL,500mM NaCl,500mM咪唑,pH8.0)进行洗脱,收集洗脱组分,用10%的SDS-PAGE结合考马斯亮蓝染色法检测分析。用Amicon Ultra超滤管将洗脱组分用镍柱结合缓冲液稀释至咪唑(imidazole)浓度小于50mM,并浓缩至3mL,加入200uL预先纯化好的TEV蛋白酶,20℃酶切过夜,用Westernblot检验酶切效率至100%。因该蛋白无法用离子交换层析纯化,所以将酶切完全后的样品浓缩至1.5mL,上样至Hiload16/60Superdex200分子筛层析柱纯化,缓冲液同镍柱结合缓冲液,需预先平衡一个柱体积,流速1mL/min,柱压设为0.3MPa,收集洗脱组分,用10%的SDS-PAGE电泳检测目的蛋白的纯度。将收集的洗脱组分浓缩至0.5mL,再上样至分析型Superdex20010/300分子筛层析柱,缓冲液同镍柱结合缓冲液,需预先平衡一个柱体积,流速0.5mL/min,柱压设为1.5MPa。结果见图3。
实施例4.HIV-1gp140蛋白的Western blot分析
将2ug纯化的去除6×His标签的gp140蛋白进行10%的SDS-PAGE电泳后,转移至PVDF膜上,以羊抗gp120多克隆抗体作为第一抗体(NIH惠赠,1∶10000稀释),辣根过氧化物酶(HRP)标记的兔抗羊IgG为第二抗体(1∶10000稀释),进行Western blot鉴定。结果见图4。
实施例5.分析型超速离心实验
沉降速率(Sedimentation velocity,SV)实验在Beckman/Coulter XL-I分析型超速离心机上进行。将待测的纯化后的gp140样品用Superdex20010/300分子筛换至超速离心缓冲液(50mM Tris,pH8.0,100mM NaCl,1mM EDTA)中,超滤浓缩。SV实验选用双通道样品池和蓝宝石窗口进行。约14.9μM纯化的gp140蛋白在4℃42000rpm进行离心沉降。实验数据用280 nm紫外检测器收集。蛋白质比重和缓冲液黏度、密度用SEDNTERP(http://www.rasmb.bbri.org/)软件计算,SV数据用Sedfit软件处理。结果见图6。
实施例6.酶联免疫吸附实验(ELASA)检测gp140的抗原反应性(antigenicity)
将4ug gp140unc和gp140分别进行一系列4倍倍比稀释(3200倍起),包被96孔酶标板,用中国疾病预防控制中心(CDC)提供的艾滋病长期不进展者的血清(1∶100稀释)作为第一抗体,HRP标记的山羊抗人IgG为第二抗体(1∶1000稀释),3,3′,5,5′-四甲基联苯胺(3,3’,5,5’-Tetramethylbenzidine,TMB)显色完成后,加入2M硫酸终止反应,在450nm波长下读板,制作标准曲线。结果见图7。
Claims (5)
1.一种针对HIV-1中国流行株CRF07的高效重组gp140免疫原的制备方法。其特征在于针对中国最广泛的流行株CRF07,并且是在结构基础上重新设计的。获得的gp140均一性好,抗原反应性高,非常适合作为免疫原用于艾滋病亚单位疫苗或者是多价联合疫苗的研发。
2.根据权利要求1所述的潜在的gp140免疫原的制备方法,其特征在于载取的gp120的片段为44-493位氨基酸(参照HIV-1HXB2标准株gp160编号,下同),截取的gp41的NHR片段为534-591位氨基酸,截取的CHR片段为623-662位氨基酸。
3.根据权利要求1所述的潜在的gp140免疫原的制备方法,其特征在于连接gp41NHR和CHR的linker为GGSGG连接,连接gp120与gp41的linker为GSGAG。
4.根据权利要求1所述的潜在的gp140免疫原的制备方法,其特征在于通过将pMT载体的V5表位替换为烟草蚀纹病毒蛋白酶(TEV酶)的酶切位点,使我们能够用TEV酶除去目的蛋白羧基末端的组氨酸标签(6×His-tag),使目的蛋白羧基末端尽可能少地带有非必要的氨基酸残基,更适合用作免疫原。
5.根据权利要求1所述的潜在的gp140免疫原的制备方法,其特征在于用重叠延伸PCR技术获得该gp140的基因片段,将目的基因克隆入真核表达载体pMT,经质粒大提去除内毒素后,与抗性筛选质粒pCoBlast一起共转染黑腹果蝇Schneider2(S2)细胞,用杀稻瘟菌素(Blasticidin S)进行阳性筛选,筛选出稳定高效分泌表达gp140的S2细胞系。扩大培养后,经镍柱亲和层析和凝胶过滤层析两步纯化,即可获得高纯度的gp140。
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