CN110478478A - 一种问号钩端螺旋体map疫苗的制备方法 - Google Patents
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Abstract
本发明公开了通过构建问号钩端螺旋体LipL21、LipL32、groEL和loa22蛋白的优势T‑和B‑细胞T‑B联合多价抗原表位融合基因及其原核表达系统，表达由抗原表位组成的多抗原肽（multiple antigenic peptide，MAP）与高分子核心载体人工交联制备MAP疫苗。本发明制备的MAP疫苗免疫效果好、成本较低，能有效诱导细胞免疫，减少了疫苗毒副作用，提高了安全性，增强了免疫针对性。
Description
技术领域
本发明属于细胞免疫技术领域，具体涉及一种问号钩端螺旋体MAP疫苗的制备方法。
背景技术
致病性钩端螺旋体（以下简称钩体）感染可引起的钩体病是全球流行的重要自然疫源性人兽共患传染病[2]。钩体病不仅是纳入国家级监控及计划免疫项目的13种传染病之一，也是自然灾害时我国重点监控和防疫的4种传染病之一。接种疫苗是预防和控制传染病最为有效和经济的手段。然而钩体具有血清群、型众多的特点，而且不同地区优势流行血清群有明显差异，不同血清群间交叉保护作用较弱的特点。因此目前使用当地流行的3-7个优势血清群（型）制备的多价全菌死疫苗不仅副作用大，而且对疫苗中未包含的其他致病性钩体感染无明显的保护作用。
近年已发现钩体含有一些属特异性蛋白抗原LipL32、OmpLl和Lipl2l用于制备通用型基因工程疫苗，虽然基因工程疫苗明显优于传统疫苗，但亦存在免疫效果差、抗原单一且成本较高的问题。病原微生物蛋白抗原分子中抗原表位（epitope）与高分子核心载体人工交联而成的多抗原肽（multiple antigenic peptide，MAP）疫苗可弥补了通用型基因工程疫苗的一些不足，能有效诱导细胞免疫，减少了疫苗毒副作用，提高了安全性，增强了免疫针对性。
发明内容
针对现有技术中存在的问题，本发明设计的目的在于提供一种问号钩端螺旋体MAP疫苗的制备方法。
本发明通过以下技术方案加以实现：
所述的一种问号钩端螺旋体MAP疫苗的制备方法，其特征在于包括以下步骤：
1）选择钩体4种蛋白的6个表位设计T-B联合多价抗原表位融合基因，经合成后插入pET-28a表达载体构建以E. coli BL21表达宿主菌的工程菌，IPTG诱导表达，纯化处理，得到多价抗原表位rLLGL蛋白；
2）采用中空纤维超滤器收集Ni-NTA亲和层析法提纯后的T-B联合多价抗原表位rLLGL蛋白，碳二亚胺活泼酯法封闭T-B联合多价抗原表位氨基，然后通过其羧基与Poly-Asp-Lys交联，采用中空纤维超滤器收集T-B表位-Poly-Asp-Lys交联物；
3）将步骤2）收集的T-B表位-Poly-Asp-Lys交联物与氢氧化铝佐剂混合后形成多抗原肽MAP疫苗。
所述的一种问号钩端螺旋体MAP疫苗的制备方法，其特征在于步骤1）中4种单位的6个表位分别为LipL21蛋白的97-122和176-184位氨基酸构成的表位、LipL32蛋白的133-160和221-247位氨基酸构成的表位、GroEL蛋白的215-247位氨基酸构成的表位、Loa22蛋白的22-90位氨基酸构成的表位。
所述的一种问号钩端螺旋体MAP疫苗的制备方法，其特征在于步骤2）中LLGL多价表位基因核苷酸序列如SEQ ID NO.1 所示。
本发明MAP疫苗免疫金地鼠观察其对问号钩体赖株感染的保护率，MAT检测动物保护试验中存活的MAP疫苗免疫金地鼠心脏采血分离血清与我国钩体标准参考株的凝集效价。结果 成功构建了多表位融合基因的原核表达系统，表达产物的相对分子质量约为18.5×103，且主要以可溶性形式存在；rLLGL与Poly-Asp-Lys交联成rLLGL交联体，rLLGL交联体兔抗血清免疫双扩散效价为1：8，该抗血清能与我国15群的钩体标准参考株发生凝集反应，ELISA证明rLLGL交联体能检测不同群型钩体感染患者血清中的抗钩体抗体。100μg和200μg的MAP疫苗对金地鼠的免疫保护率分别为60％和90％。制备的MAP疫苗免疫金地鼠血清可不同程度地凝集各问号钩体血清群。
本发明成功构建了包含钩体LipL32、OmpLl和LipL21蛋白的优势T和B细胞联合表位基因及其原核表达系统rLLGL，交联成rLLGL交联体具有良好的抗原性和交叉免疫反应性，制备MAP疫苗可作为通用型问号钩体基因工程疫苗。
具体实施方式
以下结合具体实施例对本发明做进一步详细说明。
实施例1：表位预测及鉴定
根据我国钩体血清群型参考标准株lipL21、lipL32、groEL和loa22基因序列获得的蛋白一级结构序列，应用Propred HLA-DR binding peptide prediction-ProPred服务器和EMBOSS软件包中ANTIGENIC程序分别预测其T细胞表位和B细胞表位。将预测的表位序列克隆到噬菌体载体M13KE使其展示在PⅢ蛋白表面，纯化展示产物并进行免疫活性分析，前期研究结果表明LipL21蛋白的97-122和176-184位氨基酸构成的表位、LipL32蛋白的133-160和221-247位氨基酸构成的表位、GroEL蛋白的215-247位氨基酸构成的表位、Loa22蛋白的22-90位氨基酸构成的表位具有强免疫活性，表位序列如表1所示。本研究选择LipL21-97、LipL21-176、LipL32-133、LipL32-221、GroEL-215和Loa22-90 6个表位串联构成LLGL多价表位。
表1筛选的LipL21、LipL32、GroEL和Loa22中的免疫优势T-和B-细胞联合表位
| 表位 | 表位定位 | 氨基酸序列（N→C） |
| LipL21-97 | 97-112 | ASDVV*KK#M*VGETVESA |
| LipL21-176 | 176-184 | DAL*VA#KAQEVS |
| LipL32-133 | 133-160 | TW*IRVERMSAI*MPDQIAKAAKAKPV#QKL |
| LipL32-221 | 221-247 | EV*KGSFVASV#G*LLFPPGIPGVSPLIHS |
| GroEL-215 | 215-247 | NDPFILIYDKK*ISSM#KDLIHILEKVAQAGKPLV* |
| Loa22-90 | 90-117 | FSEWSKTNAPVIKE*GLRKLPDSYALEI#T* |
注：*表示前面锚定MHCⅡ分子；#表示前面绑定B淋巴细胞
实施例2：基因合成及表达载体的构建
根据LLGL多价表位和组成表位氨基酸序列及表达宿主大肠杆菌偏爱密码子特性确定各表位对应核苷酸序列，各表位之间用柔性肽接头GGGGS核苷酸序列ggtggcggtggcagc连接，在LLGL多价表位编码序列的5'端为限制性内切酶位点EcoR I和起始密码子ATG，3'端依次为限制性内切酶位点BglⅡ、终止密码子TAA和BamH I，委托上海Invitrogen公司合成。LLGL多价表位基因核苷酸序列如下所示。将该合成基因片段通过BamH I和EcoR I酶切并纯化后亚克隆到大肠杆菌表达载体pET-28a中，转化大肠杆菌BL21，构建原核表达系统E.coli BL21pET-28a-LLGL。培养E.coli BL21pET-28a-LLGL工程菌提取质粒测序，LLGL多价表位基因核苷酸序列如SEQ ID NO.1 所示。
gaatccatggcgtctgatgtggtgaaaaaaatggtgggcgttaccgtggaaagcgcgggtggcggtggcagcctggtggcgaaagcgcaggaagtgagcggtggcggtggcagcacctggattcgagtggaacgaatgagcgcgattgcgcgggatcagattgcgaaagcggcgaaagcgaaacgggtgcagtttctgggtggcggtggcagcgaagtgaaaggcagctttgtggcgagcgtgggcctgctgtttccgccgggcattccgggcgtgagcccgctgattcattctggtggcggtggcagcaatgatccggaaattctgatttatgataaaaaaattagcagcatgaaagacctgcatattctggaaaaagtggcgcaggcgggcaaaccgctggtgggtggcggtggcagcttttctgaatggtctaaaaccaatgcgccggtgattaaagaaggcctgcgaaaactgccggattcttatgcgctggaaattaccagatcttaaggatcc
注：下划线为限制性内切酶位点，方框内为柔性肽接头GGGGS编码序列。
实施例3：重组蛋白的表达和纯化
挑取E.coli BL21pET-28a-LLGL单菌落接种到Kana抗性的10 m1 LB培养基中，37℃振荡培养过夜。按1：100的比例将培养液转接到卡那霉素抗性的10 ml新鲜LB培养基中，37℃振荡培养3 h，加入IPTG使其终浓度为1 mmol／L，37℃振荡培养4 h。取1.5 m1培养液离心收集菌体然后重悬于150µl PBS（pH7.4）缓冲液中进行低温超声波（300 V，10S x5）破碎细胞。4℃、12 000 r／min离心5 min后分别取上清和沉淀进行SDS-PAGE分析。将表达量高的菌株重新接种到卡那霉素抗性的500 ml LB培养基中进行大量诱导表达rLLGL。超声波破碎后用Ni-NTA树脂对重组蛋白进行纯化并测定纯化重组蛋白rLLGL的浓度，-80℃保存备用。
实施例4：rLLGL交联物制备
采用中空纤维超滤器收集Ni-NTA亲和层析法提纯后的T-B联合多价抗原表位rLLGL蛋白，碳二亚胺活泼酯法封闭T-B联合多价抗原表位氨基，然后通过其羧基与Poly-Asp-Lys交联，采用中空纤维超滤器收集T-B表位-Poly-Asp-Lys交联物。
实施例5：抗血清制备、效价测定和显微凝集试验（MAT）
将1 mg的rLLGL交联物与等量的完全弗氏佐剂混合均匀，皮下免疫家兔2周后，再每间隔1周加强免疫1次。末次免疫后10 d心脏取血并分离血清获得抗-rLLGL交联物血清，采用免疫双扩散试验检测其效价。将获得的兔抗血清用生理盐水进行倍比稀释，然后与等量新鲜培养的我国15群15型15株钩体参考标准株混合进行MAT测定，实验中采用正常兔血清作为阴性对照，具体的实验方法和结果判定参照文献。
实施例6：重组蛋白rLLGL的免疫活性分析
分别以1：1000稀释的兔抗钩体全菌血清和rLLGL交联物抗血清为一抗，1：5000稀释的辣根过氧化物酶标记的羊抗兔IgG为二抗进行Western blot分析。用1mg／ml的纯化重组蛋白包被96孔板，37℃静置1 h，然后4℃以6％的小牛血清封闭过夜；分别以1:500稀释的钩体病人血清（黄疸出血群、流感伤寒群、秋季群、澳洲群和波摩那群感染者血清各1份）为一抗，辣根过氧化物酶标记的羊抗人IgG或IgM为二抗进行ELISA分析。实验中以相同稀释度的正常人血清作为阴性对照，钩体感染者血清标本A450值≥对照组2倍为阳性。
实施例: 7 MAP疫苗免疫保护效果
上述交联物与氢氧化铝佐剂混合后形成多抗原肽（multiple antigenic peptide，MAP）疫苗。用4周龄雄性金地鼠（购自北京维通利华实验动物技术有限公司）检测MAP疫苗免疫对我国主要流行的致病性钩体问号钩体赖株保护效果。新鲜培养的12000 r／min离心15min(4℃)，沉淀的钩体用不同体积的EMJH培养液重悬，Petroff-Hausser countingchamber计数。先将30只金地鼠分为 5组，每组6只，其中4组分别腹腔注射107、 108、109或1010问号钩体赖株悬液1 ml，另一组注射等体积EMJH培养液作为对照，以确定问号钩体赖株最低致死量（MLD）。另将30只金地鼠分为3组，每组10只，其中二组分别腹股沟皮下注射100或200μg 的交联物与1 mg氢氧化铝佐剂混合成MAP疫苗免疫，另一组注射200μg BSA（Sigma）与氢氧化铝混合物作为对照组，间隔一周重复免疫一次。末次免疫后两周，用2倍MLD 致病性钩体腹腔注射进行攻击，观察7 d，记录动物死亡或存活数并计算保护率。上述实验动物饲养温度2l±2℃，相对湿度30%~7%，光照周期14／10 h。
实施例：8显微凝集试验（MAT）
采集上述动物保护试验中存活的MAP疫苗免疫金地鼠心脏采血分离血清。血清用PBS进行1:50、1:100、1:200、1:400、1:800、1:1600稀释，分别取0.1ml与等量新鲜培养的9群9型致病性钩体（问号钩体黄疸出血群赖型赖株、流感伤寒群流感伤寒型临6株、秋季群秋季型临4株、波摩那群波摩那型罗株、澳洲群澳洲型65-9株、七日热群七日热型56069株、犬群犬型林株、塞罗群乌尔夫型L183株）混合，37℃孵育l h，然后在暗视野显微镜下观察，以50％钩体被凝集的血清最高稀释度作为效价判断终点。实验中用正常金地鼠血清作为对照。
实验结果：
重组合成蛋白的表达及纯化：E.coli BL21pET-28a-LLGL工程菌提取质粒测序结果与预期完全一致。用终浓度1.0 mmoL／L的IPTG诱导E.coli BL21pET-28a-LLGL后能有效表达rLLGL。菌体超声波破碎后上清与沉淀、Ni-NTA纯化超声波破碎的菌体SDS-PAGE显示在约18.5×103处具有一条特异性条带，与重组蛋白预期的分子质量相符。
rLLGL交联物抗血清效价及MAT：免疫双扩散试验结果显示rLLGL交联物抗血清效价为1:8。以 我国15群15型15株的钩体标准参考株与兔抗-rLLGL交联物血清进行MAT检测，结果显示该抗血清可与我国15群的钩体标准参考株发生显著凝集，其MAT凝集效价见表2。
表2 rLLGL交联物兔抗血清与15株的钩体标准参考株的MAT
| 钩体标准参考株 | MAT | 钩体标准参考株 | MAT |
| 56601 | 1:160 | 56609 | 1:80 |
| 56602 | 1:40 | 56610 | 1:160 |
| 56603 | 1:80 | 56611 | 1:80 |
| 56604 | 1:20 | 56612 | 1: 40 |
| 56605 | 1:40 | 56613 | 1: 160 |
| 56606 | 1:40 | 56614 | 1: 40 |
| 56607 | 1:80 | 56615 | 1:80 |
| 56608 | 1:20 |
rLLGL交联物的免疫活性检测结果：分别以兔抗钩体全菌血清和兔抗-rLLGL交联物血清为一抗Western-blot结果显示，重组蛋白对应位置出现杂交条带，提示大肠杆菌表达的重组蛋白具有免疫活性。以不同血清型的钩体感染病人血清为一抗进行ELISA反应，发现均有免疫应答，见表3。
MAP免疫保护效果：问号钩体赖株对金地鼠MLD为108。2倍MLD问号钩体赖株攻击后，制备的MAP疫苗100或200μg免疫的金地鼠存活率分别为60%（6/10）和90%（9/10）。对照组金地鼠存活率为0。提示本研究获得的MAP疫苗有很好的免疫保护效果，见表4。
表4 MAT疫苗免疫金地鼠保护效果
MAT效价 MAP疫苗免疫金地鼠血清可不同程度地凝集各问号钩体血清群。与问号钩体黄疸出血群赖型赖株、流感伤寒群流感伤寒型临6株、秋季群秋季型临4株、波摩那群波摩那型罗株、澳洲群澳洲型65-9株、七日热群七日热型56069株、犬群犬型林株、塞罗群乌尔夫型L183株的MAT效价为1：50～1：800 ，见表5。
表5 疫苗免疫金地鼠血清MAT检测结果
序列表
<110> 杭州医学院
<120> 一种问号钩端螺旋体MAP疫苗的制备方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 519
<212> DNA
<213> 工程菌(engineering bacteria)
<400> 1
gaatccatgg cgtctgatgt ggtgaaaaaa atggtgggcg ttaccgtgga aagcgcgggt 60
ggcggtggca gcctggtggc gaaagcgcag gaagtgagcg gtggcggtgg cagcacctgg 120
attcgagtgg aacgaatgag cgcgattgcg cgggatcaga ttgcgaaagc ggcgaaagcg 180
aaacgggtgc agtttctggg tggcggtggc agcgaagtga aaggcagctt tgtggcgagc 240
gtgggcctgc tgtttccgcc gggcattccg ggcgtgagcc cgctgattca ttctggtggc 300
ggtggcagca atgatccgga aattctgatt tatgataaaa aaattagcag catgaaagac 360
ctgcatattc tggaaaaagt ggcgcaggcg ggcaaaccgc tggtgggtgg cggtggcagc 420
ttttctgaat ggtctaaaac caatgcgccg gtgattaaag aaggcctgcg aaaactgccg 480
gattcttatg cgctggaaat taccagatct taaggatcc 519
Claims (3)
1.一种问号钩端螺旋体MAP疫苗的制备方法，其特征在于包括以下步骤：
1）选择钩体4种蛋白的6个表位设计T-B联合多价抗原表位融合基因，经合成后插入pET-28a表达载体构建以E. coli BL21表达宿主菌的工程菌，IPTG诱导表达，纯化处理，得到多价抗原表位rLLGL蛋白；
2）采用中空纤维超滤器收集Ni-NTA亲和层析法提纯后的T-B联合多价抗原表位rLLGL蛋白，碳二亚胺活泼酯法封闭T-B联合多价抗原表位氨基，然后通过其羧基与Poly-Asp-Lys交联，采用中空纤维超滤器收集T-B表位-Poly-Asp-Lys交联物；
3）将步骤2）收集的T-B表位-Poly-Asp-Lys交联物与氢氧化铝佐剂混合后形成多抗原肽MAP疫苗。
2.如权利要求1所述的一种问号钩端螺旋体MAP疫苗的制备方法，其特征在于步骤1）中4种单位的6个表位分别为LipL21蛋白的97-122和176-184位氨基酸构成的表位、LipL32蛋白的133-160和221-247位氨基酸构成的表位、GroEL蛋白的215-247位氨基酸构成的表位、Loa22蛋白的22-90位氨基酸构成的表位。
3. 如权利要求1所述的一种问号钩端螺旋体MAP疫苗的制备方法，其特征在于步骤2）中LLGL多价表位基因核苷酸序列如SEQ ID NO.1 所示。
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