CN113150169A - 一种高免疫原性的多肽二价抗原蛋白及其制备方法和应用 - Google Patents
一种高免疫原性的多肽二价抗原蛋白及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种高免疫原性的多肽二价抗原蛋白及其制备方法和应用。本发明通过将两个相同的抗原肽X通过连接肽尾首连接获得的二价重组抗原蛋白;其中,所述抗原肽X为包含抗原表位的短肽,所述短肽由6‑15个氨基酸组成;所述连接肽为(AP)nA,连接方式为X‑(AP)nA‑X,其中,n=3、4、5、6或7。试验证明,本申请提供的多肽二价抗原蛋白免疫原性显著增强,无需偶联载体蛋白即可刺激动物体产生针对多肽的特异性抗体,这说明二价抗原在B细胞的激活过程中是必要的,为快速制备6×His‑Tag的单克隆抗体及类似多肽的单克隆抗体及短肽相关序列的免疫评价奠定基础。
Description
技术领域
本发明属于免疫学技术领域,具体涉及一种高免疫原性的多肽二价抗原蛋白及其制备方法和应用。
背景技术
组氨酸标签(His-tag),由六个His残基组成(HHHHHH),其分子量小,基本对目的蛋白本身特性几乎没有影响,不会改变目的蛋白本身的可溶性和生物学功能,在融合蛋白结晶后对蛋白的结构没有影响;His-Tag的免疫原性相对较低,可将纯化的蛋白直接注射入动物体内进行免疫并制备抗体;更重要的是它使蛋白质的纯化变得极为方便,根据组氨酸上的咪唑环可以与二价金属离子结合的原理,人们可以利用金属离子亲和层析技术纯化带有His标签的蛋白。因此,His-tag是最常用的一种蛋白表达标签,插入在目的蛋白的C末端或N末端均可,另外该标签也可以和其它的亲和标签一起构建双亲和标签使用,便于目的蛋白的纯化和检测。
因为His-Tag的免疫原性相对较低,以实验小鼠为宿主制备的His标签单抗的通常制备方法是:人工合成6*His多肽,必要时在末端添加偶联载体用到的特殊氨基酸。合成好此多肽后,通过化学方法将此多肽与载体蛋白偶联(如KLH、BSA、OVA等),偶联完成后,再用偶联好的全抗原免疫实验小鼠,免疫结束后杀死免疫好的小鼠,无菌条件下取脾脏,与骨髓瘤细胞进行融合,用ELISA或者其它手段进行筛选出阳性的克隆,筛选到的细胞经过克隆化后即形成稳定的细胞株,将此细胞株进行体外培养或小鼠体内诱生腹水形成,再从培养基或者腹水中纯化即可得到His标签鼠单抗。该方法操作复杂,而且由于引入外源载体蛋白,为筛选到特异性针对His-Tag的单克隆抗体带来较大困难。
目前,表位肽的研究已经在疫苗相关技术领域得到广泛应用,但其仍存在免疫原性低的缺陷;此外,其他短肽,如从7肽库、12肽库随机筛选到的7肽或12肽等,也因为免疫原性相对较低而很难刺激机体产生免疫应答,通常研究者也是通过化学方法将此类多肽与载体蛋白偶联(如KLH、BSA、OVA等),偶联完成后,再用偶联好的全抗原免疫实验小鼠,这一过程相对繁琐,为短肽类物质的免疫评价及单克隆抗体制备带来一定困难。因此,如何更高效便捷的获得具备高免疫原性的抗原蛋白是急需解决的技术问题。
发明内容
本发明的目的在于提供一种高免疫原性的多肽二价抗原蛋白。
本发明的第二个目的在于提供上述高免疫原性的多肽二价抗原蛋白的制备方法。
本发明的第三个目的在于提供上述述高免疫原性的多肽二价抗原蛋白的应用。
为了实现上述目的,本发明采用以下技术方案:
一种高免疫原性的多肽二价抗原蛋白,将两个相同的抗原肽X通过连接肽尾首连接获得的二价重组抗原蛋白;所述抗原肽X为包含抗原表位的短肽,所述短肽由6-15个氨基酸组成;所述连接肽为(AP)nA,连接方式为X-(AP)nA-X,其中,n=3、4、5、6或7。
优选的,所述抗原肽X的氨基酸序列为HHHHHH。
优选的,所述抗原肽X包含PCV3 Cap蛋白抗原表位140KHSRYFT146,其氨基酸序列为KHSRYFT;所述连接肽为(AP)5A,连接方式为KHSRYFT-(AP)5A-KHSRYFT。
优选的,所述抗原肽X包含PCV3 Cap蛋白抗原表位140KHSRYFT146,其氨基酸序列为TSKKKHSRYFTPKPI;所述连接肽为(AP)5A,连接方式为TSKKKHSRYFTPKPI-(AP)5A-TSKKKHSRYFTPKPI。
一种高免疫原性的多肽二价抗原蛋白的制备方法,将两个相同的抗原肽X通过连接肽尾首相连,获得高免疫原性的多肽二价抗原蛋白;所述抗原肽X为包含抗原表位的短肽,所述短肽由6-15个氨基酸组成;所述连接肽为(AP)nA,连接方式为X-(AP)nA-X,其中,n=3、4、5、6或7。
优选的,所述抗原肽X的氨基酸序列为HHHHHH。
优选的,所述抗原肽X包含PCV3 Cap蛋白抗原表位140KHSRYFT146,其氨基酸序列为KHSRYFT,所述连接肽为(AP)5A,连接方式为KHSRYFT-(AP)5A-KHSRYFT。
优选的,所述抗原肽X包含PCV3 Cap蛋白抗原表位140KHSRYFT146,其氨基酸序列为TSKKKHSRYFTPKPI,所述连接肽为(AP)5A,连接方式为TSKKKHSRYFTPKPI-(AP)5A-TSKKKHSRYFTPKPI。
本发明还提供了上述的高免疫原性的多肽二价抗原蛋白在制备单克隆抗体或多克隆抗体中的应用;所述多肽二价抗原蛋白经弗氏佐剂乳化后直接免疫动物,能够刺激动物体产生针对多肽二价抗原蛋白的特异性抗体。
本发明取得的有益效果:
本发明提供的高免疫原性的多肽二价抗原蛋白及其制备方法。多肽二价抗原蛋白是将两个相同的抗原肽X通过连接肽尾首连接获得的二价重组抗原蛋白;其中,所述抗原肽X为包含抗原表位的短肽,所述短肽由6-15个氨基酸组成;所述连接肽为(AP)nA,连接方式为X-(AP)nA-X,其中,n=3、4、5、6或7。试验证明,本申请提供的多肽二价抗原蛋白免疫原性显著增强,无需偶联载体蛋白即可刺激动物体产生针对多肽的特异性抗体,这说明二价抗原在B细胞的激活过程中是必要的,为快速制备6×His-Tag的单克隆抗体及类似多肽的单克隆抗体及短肽相关序列的免疫评价奠定基础。
附图说明
图1是多肽质谱(MS)鉴定结果1;
图2是多肽质谱(MS)鉴定结果2;
图3是高效液相色谱(HPLC)鉴定结果1;
图4是高效液相色谱(HPLC)鉴定结果2;
图5是二价抗原免疫机体后不同时间刺激机体产生抗体的水平;
图6是二价抗原免疫后刺激机体产生抗体的差异;
图7是Dot-ELISA鉴定。
其中,图中A为His单抗鉴定结果:1为H6-(AP)5A-H6;2为KHSRYFT-(AP)5A-KHSRYFT;图中B为PCV3 Cap蛋白单抗鉴定结果:1为KHSRYFT-(AP)5A-KHSRYFT;2为H6-(AP)5A-H6;图中C为PCV3 Cap蛋白单抗鉴定结果:1为TSKKKHSRYFTPKPI-(AP)5A-TSKKKHSRYFTPKPI;2为H6-(AP)5A-H6。
具体实施方式
下面结合具体实施方式对本发明作进一步描述,但本发明的保护范围并不仅限于此;本发明所使用的仪器设备如无特别说明,均为市售常规仪器设备;所用试剂材料,如无特别说明,均为市售常规试剂材料;重多肽合成等技术服务由上海生工生物工程有限公司提供。
实施例1
一、材料与方法
1.1多肽合成
所涉及多肽包括:
(1)一条用以偶联卵清蛋白OVA的6×His-Tag CH6,用以作为多肽免疫小鼠抗6×His-Tag效价评定的ELISA包被原。
(2)一条以两个6×His-Tag直接串联的H6-H6;两条分别以一个6×His-Tag与刚性15肽linker(AP)7A的N或C端相连接的H6-(AP)7A、(AP)7A-H6;以及一条以两个6×His-Tag同时连接于刚性15肽linker(AP)7A两端的H6-(AP)7A-H6。该组多肽用以研究二价抗原在B细胞激活过程中的必要性。
(3)一组多肽,分别以两个6×His-Tag同时连接于长度依次递减的刚性linker,包括了linker长度分别为13aa、11aa、9aa、7aa、5aa、3aa、1aa的H6-(AP)6A-H6、H6-(AP)5A-H6、H6-(AP)4A-H6、H6-(AP)3A-H6、H6-(AP)2A-H6、H6-APA-H6、H6-A-H6。该组linker用以研究二价抗原间需要的最短距离,以避免两个BCR因为空间位阻效应导致的交联失败。
本发明所涉及的所有多肽均由吉尔生化(上海)有限公司合成,表1是多肽名称序列及纯度。
表1多肽序列及纯度
名称 | 氨基酸序列 | 纯度 |
H<sub>6</sub>-H<sub>6</sub> | HHHHHHHHHHHH | 98.14% |
H<sub>6</sub>-(AP)<sub>7</sub>A | HHHHHHAPAPAPAPAPAPAPA | 97.49% |
(AP)<sub>7</sub>A-H<sub>6</sub> | APAPAPAPAPAPAPAHHHHHH | 92.79% |
H<sub>6</sub>-(AP)<sub>7</sub>A-H<sub>6</sub> | HHHHHHAPAPAPAPAPAPAPAHHHHHH | 91.91% |
H<sub>6</sub>-(AP)<sub>6</sub>A-H<sub>6</sub> | HHHHHHAPAPAPAPAPAPAHHHHHH | 91.20% |
H<sub>6</sub>-(AP)<sub>5</sub>A-H<sub>6</sub> | HHHHHHAPAPAPAPAPAHHHHHH | 93.82% |
H<sub>6</sub>-(AP)<sub>4</sub>A-H<sub>6</sub> | HHHHHHAPAPAPAPAHHHHHH | 90.53% |
H<sub>6</sub>-(AP)<sub>3</sub>A-H<sub>6</sub> | HHHHHHAPAPAPAHHHHHH | 90.97% |
H<sub>6</sub>-(AP)<sub>2</sub>A-H<sub>6</sub> | HHHHHHAPAPAHHHHHH | 91.83% |
H<sub>6</sub>-APA-H<sub>6</sub> | HHHHHHAPAHHHHHH | 98.67% |
H<sub>6</sub>-A-H<sub>6</sub> | HHHHHHAHHHHHH | 90.74% |
CH<sub>6</sub> | CHHHHHH | 90.96% |
1.2多肽的鉴定
本发明所涉及的所有多肽由质谱(MS)和高效液相色谱(HPLC)鉴定,鉴定结果分别如图1-2、图3-4所示。
1.3小鼠免疫实验
免疫用多肽干粉以超纯水直接溶解,至终浓度为2mg/mL。多肽水溶液与弗式完全佐剂以体积比1:1混合均匀,并超声乳化后备用;取6至8周龄BALC/c雌性小鼠随机分组后,采取背部皮下多点注射将乳化多肽进行免疫,每只小鼠免疫剂量为100μg。其中,H6-H6、H6-(AP)7A、(AP)7A-H6、H6-(AP)7A-H6组每条多肽免疫8只小鼠,并在免疫后0d、3d、7d、10d、14d、17d、21d断尾采血,分离血清-40℃保存备用;H6-(AP)6A-H6、H6-(AP)5A-H6、H6-(AP)4A-H6、H6-(AP)3A-H6、H6-(AP)2A-H6、H6-APA-H6、H6-A-H6每条多肽免疫3只小鼠,并在免疫后21d断尾采血,分离血清-40℃保存备用。
1.4OVA偶联CH6
(1)取4mg OVA溶于500μL偶联buffer(0.1M PB,pH 7.2,0.15M NaCl,1μM EDTA);(2)取1mg Sulfo-SMCC溶于50μL DMSO,与OVA溶液混合,室温静置1h;
(3)4℃条件下,将混合液对偶联buffer过夜透析,换液3次去除多余Sulfo-SMCC后调整活化OVA溶液浓度为5mg/mL;
(4)取4mg多肽CH6,溶于400μL含5mM EDTA的0.01M PB buffer,制备为10mg/mL的多肽储存液;
(5)取10μL多肽储存液与10μL含5mM EDTA的0.01M PB buffer混匀,并加入活化OVA溶液20μL,室温反应4h后,置于4℃条件下过夜孵育
(6)以0.01M PB调整多肽浓度至1mg/mL,作为ELISA包被原于-20℃分装保存。1.5anti-6×His-Tag抗体间接ELISA
(1)CBS稀释包被原OVA-CH6至2μg/mL,50μL/孔包被于96孔板,37℃条件下包被2h;(2)以5%脱脂奶(PBST,0.05%tween-20),与37℃条件下封闭1h;
(3)鼠血清:5%脱脂奶(PBST,0.05%tween-20)=1:50稀释,50μL/孔加于96孔板,37℃条件下孵育1h;
(4)PBST洗板6次,HRP标记羊抗鼠二抗1:1000倍稀释后,50μL/孔加于96孔板,37℃条件下孵育1h;
(5)PBST洗板6次,室温条件下TMB显色10min后,以2M H2SO4终止反应;于多功能酶标仪读取OD450nm数值。
二、结果
2.1二价抗原以及氨基酸免疫抗体消长规律分析。
如图5所示,多肽H6-(AP)7A以及(AP)7A-H6免疫小鼠免疫后21d内均没有产生anti-6×His-Tag抗体。表明单独将一个6×His-Tag连接于linker并未引起B细胞的激活;多肽H6-(AP)7A-H6免疫小鼠在免疫10d后,血清中anti-6×His-Tag抗体持续升高。这表明通过将两个6×His-Tag表位同时连接于15肽linker(AP)7A可能有利于两个6×His-Tag通过有效地交联BCR,使B细胞能够激活,并产生特异性anti-6×His-Tag抗体;这说明二价抗原在B细胞的激活过程中是必要的;其次,同样是二价的H6-H6,小鼠在免疫后的21d内同样未产生anti-6×His-Tag抗体,这表明H6之间需要一定的氨基酸数量,以利于抗原分子对BCR的有效交联。
2.2二价抗原以及氨基酸免疫免疫效价测定。
如图6所示,进一步的,通过将两个6×His-Tag同时连接于1-13肽linker,免疫后21d的ELISA结果表明,至少7aa的linker(H6-(AP)3A-H6)对于免疫小鼠产生anti-6×His-Tag抗体是必要的,7aa~13aa的linker(H6-(AP)3A-H6)表明两个相同的抗原表位通过7aalinker连接可以激起机体特异性的免疫反应。
实施例2
Dot-ELISA鉴定二价抗原X-(AP)nA-X(其中n=3,4,5,6或7)与相应单克隆抗体的反应性
1.二价抗原与相应单克隆抗体的反应性
Dot-ELISA即斑点酶联免疫吸附测定法具体操作步骤如下:
1)压印:剪取一片合适大小的NC膜,用200μl吸头的圆形底座在膜的表面按顺序压印,以此作为点样部位。
2)点样:用2.5μl Eppendorf微量移液枪分别吸取0.5~1μl待检二价抗原(A:H6-(AP)5A-H6、B:KHSRYFT-(AP)5A-KHSRYFT或C:TSKKKHSRYFTPKPI-(AP)5A-TSKKKHSRYFTPKPI),滴于圆圈内,自然干燥。二价抗原A组选择B组多肽作为NC对照,二价抗原B、C组均选择A组多肽H6-(AP)5A-H6作为NC对照。
3)封闭:每孔加入50μl的5%脱脂奶,37℃封闭1h;PBST洗板3次;
4)加一抗(A组为商品化His单抗;B、C组选用实验室制备的可以特异性识别PCV3Cap蛋白KHSRYFT的单克隆抗体17H10):1:5000稀释,37℃孵育30min;
5)洗涤:PBST洗6次;
6)加酶标二抗:将HRP标记的羊抗鼠IgG(1:1000)用PBS稀释至工作浓度后,37℃孵育30min;
7)洗涤:PBST洗板6次;
8)显色:取AEC显色液,按所需的量混匀后显色3min,观察并记录实验结果。
2.实验结果
Dot-ELISA实验结果显示(图7),商品化His单抗可以与二价抗原H6-(AP)5A-H6与发生特异性反应,而与二价抗原KHSRYFT-(AP)5A-KHSRYFT不反应;单克隆抗体17H10可以识别二价抗原KHSRYFT-(AP)5A-KHSRYFT以及TSKKKHSRYFTPKPI-(AP)5A-TSKKKHSRYFTPKPI,而与二价抗原H6-(AP)5A-H6不发生反应。
<110> 河南中泽生物工程有限公司
<120> 一种高免疫原性的多肽二价抗原蛋白及其制备方法和应用
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<170> PatentIn version 3.5
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Claims (9)
1.一种高免疫原性的多肽二价抗原蛋白,其特征在于,将两个相同的抗原肽X通过连接肽尾首连接获得的二价重组抗原蛋白;所述抗原肽X为包含抗原表位的短肽,所述短肽由6-15个氨基酸组成;所述连接肽为(AP)nA,连接方式为X-(AP)nA-X,其中,n=3、4、5、6或7。
2.根据权利要求1所述多肽二价抗原蛋白,其特征在于,所述抗原肽X的氨基酸序列为HHHHHH。
3.根据权利要求1所述的多肽二价抗原蛋白,其特征在于,所述抗原肽X包含PCV3 Cap蛋白抗原表位140KHSRYFT146,其氨基酸序列为KHSRYFT;所述连接肽为(AP)5A,连接方式为KHSRYFT-(AP)5A-KHSRYFT。
4.根据权利要求1所述的多肽二价抗原蛋白,其特征在于,所述抗原肽X包含PCV3 Cap蛋白抗原表位140KHSRYFT146,其氨基酸序列为TSKKKHSRYFTPKPI;所述连接肽为(AP)5A,连接方式为TSKKKHSRYFTPKPI-(AP)5A-TSKKKHSRYFTPKPI。
5.一种高免疫原性的多肽二价抗原蛋白的制备方法,其特征在于,将两个相同的抗原肽X通过连接肽尾首相连,获得高免疫原性的多肽二价抗原蛋白;所述抗原肽X为包含抗原表位的短肽,所述短肽由6-15个氨基酸组成;所述连接肽为(AP)nA,连接方式为X-(AP)nA-X,其中,n=3、4、5、6或7。
6.根据权利要求5所述的制备方法,其特征在于,所述抗原肽X的氨基酸序列为HHHHHH。
7.根据权利要求5所述的制备方法,其特征在于,所述抗原肽X包含PCV3 Cap蛋白抗原表位140KHSRYFT146,其氨基酸序列为KHSRYFT,所述连接肽为(AP)5A,连接方式为KHSRYFT-(AP)5A-KHSRYFT。
8.根据权利要求5所述的制备方法,其特征在于,所述抗原肽X包含PCV3 Cap蛋白抗原表位140KHSRYFT146,其氨基酸序列为TSKKKHSRYFTPKPI,所述连接肽为(AP)5A,连接方式为TSKKKHSRYFTPKPI-(AP)5A-TSKKKHSRYFTPKPI。
9.如权利要求1-4任一项所述的高免疫原性的多肽二价抗原蛋白在制备单克隆抗体或多克隆抗体中的应用,其特征在于,所述多肽二价抗原蛋白经弗氏佐剂乳化后直接免疫动物,能够刺激动物体产生针对多肽二价抗原蛋白的特异性抗体。
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