CN111529700A - 一种多房棘球蚴亮氨酰胺肽酶亚单位疫苗lap及其制备方法与应用 - Google Patents
一种多房棘球蚴亮氨酰胺肽酶亚单位疫苗lap及其制备方法与应用 Download PDFInfo
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- CN111529700A CN111529700A CN202010418240.9A CN202010418240A CN111529700A CN 111529700 A CN111529700 A CN 111529700A CN 202010418240 A CN202010418240 A CN 202010418240A CN 111529700 A CN111529700 A CN 111529700A
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- echinococcus multilocularis
- subunit vaccine
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Abstract
本发明公开了一种多房棘球蚴亮氨酰胺肽酶亚单位疫苗LAP及其制备方法与应用,亚单位疫苗LAP的活性成分是一条多肽,主要由多房棘球蚴抗原蛋白LAP氨基酸序列构成。本发明主要通过基因合成技术合成多房棘球蚴亚单位疫苗LAP基因序列,通过双酶切连接至表达载体中,然后将表达载体转化进Arctic Express进行融合蛋白的表达。蛋白纯化后,获得多房棘球蚴亚单位疫苗LAP。该多房棘球蚴亚单位疫苗能够诱发机体产生针对多房棘球蚴T细胞及B细胞免疫应答和高滴度特异性抗体体液免疫应答,能有效预防小鼠感染多房棘球蚴,可用于预防和治疗多房棘球蚴感染相关性疾病。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及到一种多房棘球蚴亚单位疫苗亮氨酰胺肽酶LAP的应用及制备方法。
背景技术
多房棘球蚴感染人称为泡型包虫病(alveolar echinococcosis,AE),该疾病是一种人畜共患的寄生虫疾病。2012年至2016年期间,一项针对中国包虫病的全国性调查显示,西部9个省份约5000万人面临感染风险,近17万人感染包虫病。多房棘球蚴大部分的原发病灶都在肝脏,大部分包虫病患者就诊较晚。早期的包虫病不会引起症状,AE病变可保持无症状10至15年。临床症状通常出现在肝内囊肿直径超过10cm后,或超过70%的器官体积被囊肿或囊肿占据,导致胆管、肝静脉、门静脉或肝动脉受到物理压迫或损伤。目前棘球蚴病的治疗多采用苯并咪唑(阿苯达唑或甲苯达唑),但疗程长、副作用大,患者依从性较差。调查统计包虫病患者虽可通过手术治愈但术后仍有复发风险,部分患者需长期服药或再次手术,危害民众身心健康的同时也会影响畜牧业的正常运转。根据棘球蚴病人畜共患病特点,采用疫苗的方式可能是控制传染病最有效的手段。
发明内容
本发明针对现有技术存在的问题,第一个目的是一种针对多房棘球蚴亮氨酰胺肽酶的亚单位疫苗。
本发明第二个目的是提供多房棘球蚴亮氨酰胺肽酶亚单位疫苗的制备方法。
本发明第三个目的是提供多房棘球蚴亮氨酰胺肽酶亚单位疫苗的用途。
为了解决上述技术问题,本发明采用如下技术方案:一种多房棘球蚴亮氨酰胺肽酶亚单位疫苗LAP,其特征在于:其活性成分是一条多肽,其氨基酸序列如序列1所示。
其核苷酸序列如序列2所示。
一种表达载体,其特征在于:包含前述的核苷酸序列。
一种转基因细胞系,其特征在于:包含前述的核苷酸序列。
一种宿主菌,其特征在于:包含前述的核苷酸序列。
该多房棘球蚴亮氨酰胺肽酶亚单位疫苗LAP的制备方法,通过基因合成技术合成多房棘球蚴多表位肽LAP的核苷酸序列,构建含有融合基因LAP的重组表达载体pCzn1-LAP及其重组基因工程菌株Arctic Express;重组基因工程菌株Arctic Express发酵后,经Ni-IDA镍离子交换层析纯化,获得该疫苗的融合蛋白LAP。
其技术路线详述如下:
重组表达质粒pCzn1-LAP(含有融合基因LAP)的构建。
采用基于PAS(PCR-based Accurate Synthesis)的方法合成基因LAP,通过双酶切连接至pCzn1载体的Nde I和Xba I之间,获得的重组质粒pCzn1-LAP。
融合蛋白LAP的原核表达及纯化。
将重组表达载体pCzn1-LAP转化进大肠杆菌Arctic Express中,构建重组基因工程菌株Arctic Express/pCzn1-LAP。利用IPTG诱导表达,并通过Ni-IDA镍离子亲和层析获得电泳纯度的融合蛋白LAP,即为多房棘球蚴亚单位疫苗LAP。
该多房棘球蚴亮氨酰胺肽酶亚单位疫苗LAP能够激发机体产生针对多房棘球蚴B细胞及T细胞的免疫应答。
该多房棘球蚴亮氨酰胺肽酶亚单位疫苗LAP还包括在制备预防和治疗多房棘球蚴感染的药物组合中的应用。可用于预防和治疗多房棘球蚴感染相关疾病。
本发明采用多房棘球蚴抗原性较高的抗原亮氨酰氨肽酶(leucylaminopeptidase,LAP),亮氨酸氨基肽酶(LAP)是一种金属肽酶,参与外源蛋白的分解代谢,在寄生虫和微生物中是蛋白质转化和成熟的最后步骤所必需的,在肽能的转换中起着关键作用。这类氨肽酶抑制剂对恶性疟原虫、布鲁氏锥虫、肝片吸虫等均具有较好的抑制作用。LAP在多种寄生虫和微生物中存在,具有良好的免疫原性,且在不同寄生虫中氨基酸序列差异性较大。LAP在多房棘球蚴和细粒棘球蚴中序列相似度62.63%,与小鼠、犬相似度14.8%,与人源无相似序列,因此LAP可作为多房棘球蚴疫苗抗原蛋白。
本发明采用分子克隆技术将LAP核苷酸序列经大肠杆菌密码子优化后克隆到质粒pCzn1中,经原核系统表达并纯化获得重组蛋白LAP;继而将亚单位疫苗LAP免疫小鼠后经ELISA、小鼠脾淋巴细胞增殖实验等技术考察亚单位疫苗LAP的免疫原性及生物活性;最后结合多房棘球蚴小鼠继发感染模型评价LAP预防效果。
本发明具有以下优点:1、亮氨酰胺肽酶是多房棘球蚴总蛋白中与泡型包虫病病人血清免疫反应较强的部分可溶性蛋白,具有良好的抗原性。2、多房棘球蚴亚单位疫苗亮氨酰胺肽酶与人源无相似序列,与小鼠、犬相似度较低,不会引起自身免疫攻击。3、多房棘球蚴亚单位基因工程疫苗亮氨酰胺肽酶,安全性高、产量高、纯度高,稳定性强。4、亮氨酰胺肽酶具有较强的免疫原性,能够诱发针对多房棘球蚴抗原蛋白亮氨酰胺肽酶及总蛋白高滴度的特异性抗体产生。5、能有效预防小鼠感染多房棘球蚴。
附图说明
图1:重组表达载体pCzn1-LAP的双酶切鉴定。泳道1:pCzn1-LAP酶切前质粒;泳道2:pCzn1-LAP/Nde I+Xba I;泳道M:DNA Marker
图2:重组表达载体pCzn1-LAP载体构建图谱。
图3:多房棘球蚴多表位肽融合蛋白LAP的原核表达。泳道M:蛋白质Marker;泳道1:未加IPTG诱导菌液蛋白;泳道2:加IPTG诱导后菌液蛋白;泳道3:加入0.5mM IPTG 11℃诱导后菌液离心后上清;泳道4:加入0.5mM IPTG 11℃诱导菌液离心后沉淀。
图4:多房棘球蚴多表位肽融合蛋白LAP的Ni-IDA亲和层析纯化。泳道M:蛋白质Marker;泳道1:LAP未纯化蛋白;泳道2:20mM咪唑洗脱的杂蛋白和部分目的蛋白;泳道3:纯化后的LAP蛋白样品。
图5:多房棘球蚴亚单位疫苗LAP诱发抗LAP IgG抗体的检测。多房棘球蚴亚单位疫苗LAP能诱发产生较高滴度抗LAP的IgG抗体,并具有较高的抗体效价。
图6:多房棘球蚴亚单位疫苗LAP诱发抗多房棘球蚴总蛋白IgG抗体的检测。多房棘球蚴亚单位疫苗LAP能够产生一定滴度抗多房棘球蚴总蛋白的IgG抗体。
图7:多房棘球蚴亚单位疫苗LAP致敏小鼠脾脏淋巴细胞对抗原刺激的增殖反应。
图8:多房棘球蚴亚单位疫苗LAP预防效果。
图9:多房棘球蚴亚单位疫苗LAP预防后攻毒4个月囊泡重量。
图10:多房棘球蚴亚单位疫苗LAP预防后攻毒4个月囊泡数量。
具体实施方式
下面将结合附图对本发明的技术方案进行清楚、完整的描述,当然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例,并不意味着对本发明的任何限制。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
材料
1、IPTG溶液:称取2.4g IPTG溶于10mL无菌水,用0.22μm滤器过滤除菌,小份分装,-20℃保存。
2、氨苄青霉素(Amp)贮液(100mg/mL):称取1g氨苄青霉素(Amp)溶于10mL无菌水,制得浓度为100mg/mL的贮存液,通过0.22μm细菌滤器过滤除菌,溶液分装保存于-20℃冰箱中。
3、培养基:(1)LB液体培养基:称取10g胰蛋白胨、5g酵母提取物和5g NaCl,加蒸馏水至1L,调整pH至7.4,高压蒸气灭菌。(2)LB固体培养基:1.5g琼脂粉/100mL LB培养液,高压灭菌后,倾倒平板。
4、DNA电泳缓冲液(50xTAE):称取242g Tris、37.2g Na2EDTA·2H2O和57.1ml冰乙酸,加水至1L,使用时稀释50倍。
5、SDS-PAGE电泳缓冲液(5X):称取Tris粉末15.1g、甘氨酸94g、SDS 5.0g;加入约800mL的去离子水,搅拌溶解;加去离子水定容至1L,室温保存;注意:加水时应让水延着壁缓缓流下,以避免由于SDS的原因产生很多泡沫。
6、考马斯亮蓝蛋白染色试剂:(1)考马斯亮蓝G-250染液(蛋白质定量用):考马斯亮蓝G-250 100mg溶解在50mL 95%乙醇中,然后加入86%磷酸100mL,用蒸馏水稀释至1000mL。(2)脱色液:250mL乙醇、80mL冰醋酸用蒸馏水稀释至1000mL。
8、实验动物:ICR小鼠:为SPF级,雄性,6~8周龄,购自西安交通大学医学部实验动物中心,许可证号:SCXK(陕)2018-001。
9、ELISA试剂:(1)包被液:1.6g Na2CO3,2.9g NaHCO3,0.2g NaN3,加双蒸水至1L,调pH值至9.6。(2)洗涤液:分别称取0.2g KH2PO4,2.9g Na2HPO4·12H2O,8.0g NaCl,0.2gKCl,0.5ml Tween-20,加入ddH2O定容至1L(PBST)。(3)封闭液:称取3.0g BSA溶解于100ml洗涤缓冲液中,过滤除菌后4℃保存。(4)底物液:可溶性单组份TMB底物溶液。(5)终止液:量取蒸馏水178.3mL,逐滴加入浓硫酸21.7mL(1M H2SO4)。
10、淋巴细胞增殖实验主要试剂(1)小鼠脾脏淋巴细胞分离液(购于CEDARLANE公司,CL5031)(2)RPMI-1640完全培养液:在RPMI-1640基础培养液加入10%胎牛血清、100U/mL青霉素、100μg/mL链霉素。(3)RPMI-1640不完全培养液:称取10.4g RPMI-1640干粉、2.4gHEPES、0.75g NaHCO3,加去离子水至1L,pH 7.4,超滤除菌,分装。
实施例1:重组表达载体pCzn1-LAP(含有融合基因LAP)的构建
将LAP的氨基酸序列按照大肠杆菌密码子偏爱性原则转化成相应的核苷酸序列,采用基于PAS(PCR-based Accurate Synthesis)的方法,设计全长拼接引物,在引物的两端各设计了保护性碱基合成基因LAP,通过克隆位点Nde I和Xba I连入表达载体pCzn1。
结果:利用Nde I和Xba I双酶切待检重组质粒pCzn1-LAP,37℃反应2h,用1%的琼脂糖凝胶电泳检测,发现双酶切的DNA片段约为1700bp,与融合基因LAP的理论大小一致,如(图1)所示。重组表达载体pCzn1-LAP的载体构建图谱如(图2)所示。将获得的重组质粒pCzn1-LAP转入TOP10克隆菌株,挑取阳性克隆子测序,测序结果与预期序列完全一致,且无移码突变。
实施例2:多表位肽融合蛋白LAP的原核表达
将验证正确的重组表达质粒pCzn1-LAP转入到大肠杆菌Arctic Express菌株中。在预先制备好的含50μg/mL Amp的LB平板上,接种环划线基因工程菌株pCzn1-LAP/ArcticExpress,倒置于37℃培养箱,过夜培养后,挑取单个菌落,接种于含50μg/mL Amp的LB培养基中,37℃,220rpm,培养过夜。以2%接种量分别接种重组菌于含50μg/mL Amp LB培养基中,37℃,220rpm,培养至至菌体OD600为0.6-0.8(约2h),加入IPTG使终浓度达到1mmol/L,37℃,220rpm诱导表达4h,以未加IPTG诱导的载体菌pCzn1-LAP/Arctic Express作为阴性对照。
结果:与对照菌株对比,基因工程重组菌株pCzn1-LAP/Arctic Express在约57KD处出现目的蛋白条带,与多表位肽融合蛋白LAP的理论大小相符合(图3)。多表位肽融合蛋白LAP在包涵体蛋白中存在。
实施例4:多表位肽融合蛋白LAP的纯化
(1)包涵体蛋白的变复性
将菌体沉淀重悬于20ml lysis buffer(20mM Tris-HCl containing 1mM PMSFand bacteria protease inhibitor cocktail,pH 8.0),超声破碎(功率400W,工作4sec,间歇8sec,共20min);将超声破碎的细胞裂解液4℃10000g离心20min,收集沉淀;使用包涵体洗涤液(20mM Tris,1mM EDTA,2M尿素,1M NaCl,1%Triton X-100,pH8.0)洗涤包涵体3次;用溶解缓冲液(20mM Tris,5mM DTT,8M尿素pH8.0),按一定比例溶解包涵体,4℃放置过夜;室温,15000rpm离心15min;将上述溶液滴加20mM Tris-HCl 5mM EDTA Buffer PH7.8缓冲液中,逐步成倍梯度稀释缓慢搅拌,将蛋白溶液装入透析袋于PBS pH7.4溶液中透析过夜。
(2)Ni-IDA镍离子亲和层析柱的纯化
利用低压层析系统,蛋白溶液以0.5ml/min流速上样至Ni-IDA Binding-Buffer预平衡的Ni-IDA-Sepharose CL-6B亲和层析柱;用Ni-IDA Binding-Buffer以0.5mL/min流速冲洗,至流出液OD280值到达基线;用Ni-IDA Washing-Buffer(20mM Tris-HCl,20mM咪唑,0.15M NaCl,pH8.0)以1mL/min流速冲洗,至流出液OD280值到达基线;用Ni-IDA Elution-Buffer(20mM Tris-HCl,250mM咪唑,0.15M NaCl,pH8.0)以1mL/min流速洗脱目的蛋白,收集流出液;上述收集的蛋白溶液加入透析袋中,使用PBS(PH7.4)进行透析过夜。
结果:经Ni-IDA镍离子亲和层析柱的纯化后,收集的各个蛋白峰进行SDS-PAGE分析,可以发现目的蛋白集中在250mM咪唑洗脱产生的蛋白峰。通过凝胶成像检测仪分析在250mM咪唑洗脱的目的蛋白纯度可达电泳纯(图4)。
实施例5:亚单位疫苗LAP的免疫原性和免疫特异性研究
(1)ICR小鼠的免疫
实验分组:将SPF级ICR小鼠随机分为3组,分别为多表位融合蛋白LAP免疫组、和PBS免疫组。每组6只ICR小鼠,共18只,详细分组如表1所示:
表1:SPF级ICR小鼠分组及免疫方案
免疫方式:用75%酒精对小鼠腹部消毒,然后腹部多点皮下注射表位融合蛋白LAP和弗氏完全佐剂的混合乳化剂;每隔一周加强免疫一次,第2、3周加弗氏不完全佐剂,第4周直接注射表位融合蛋白溶液加强免疫。
抗血清的采集:在末次免疫后第五天,摘小鼠眼球采血,收集血液,放置待血清完全分离,3000rpm离心5分钟分装血清,-80℃冻存备用。
(2)抗血清中特异性抗体的ELISA检测
将抗原(LAP和多房棘球蚴总蛋白)分别用包被液稀释成10μg/mL,100μl/孔包被ELISA板,4℃过夜。用洗涤液洗4次后,每孔加入300μl封闭液,37℃封闭2h。用洗涤液洗4次后,将抗血清(鼠抗LAP抗血清和鼠抗rLTB抗血清)和小鼠阴性血清倍比稀释后加入ELISA板,100μL/孔,在37℃下孵育60min。用洗涤液洗4次后,加入HRP标记的羊抗鼠IgG(1:10000),100μL/孔,在37℃下孵育1h。用洗涤液洗4次后,加入100μL/孔TMB底物显色液,室温避光反应10min,加50μL终止液终止反应。用酶标仪测定各孔OD450值。
结果:LAP的包被浓度为10μg/mL,通过ELISA检测,多房棘球蚴亚单位疫苗LAP能够产生针对LAP的特异性IgG抗体,而PBS免疫组不能产生抗LAP抗体(图5);多房棘球蚴总蛋白的包被浓度为10μg/ml,通过ELISA检测,亚单位疫苗LAP能够产生抗多房棘球蚴总蛋白的抗体,而PBS免疫组不能产生抗多房棘球蚴总蛋白的特异性IgG抗体(图6)。
(3)小鼠脾脏淋巴细胞增殖实验
将经抗原免疫的小鼠脱臼处死,泡75%酒精5min后,移入超净工作台。用剪刀小心剪开小鼠的腹部外皮,再剪开小鼠的腹腔,用镊子取出小鼠脾脏(暗红色)。在小平皿中放入2-3mL Lympholyte淋巴细胞分离液;用镊子固定尼龙网,然后用注射器活塞轻轻研磨小鼠脾脏,使得分散的单细胞透过尼龙网进入淋巴细胞分离液中;把悬有脾脏细胞的分离液立即转移到离心管中,离心前再覆盖上大约1mL的1640培养基;1000g-1500g离心20min。离心结束后淋巴细胞会在1640培养基下层悬浮。用含10%小牛血清的RPMI-1640培养基制备成一定浓度的细胞悬液(5x105/mL)。在96孔平底培养板中,每孔加入100μL细胞,同时加入LAP抗原蛋白20μg/ml、LAP抗原表位肽(LAP79-93和LAP106-120)和生理盐水各100μL,终体积为200μl/孔,每组做3个平行孔。将加好的96孔平底培养板放置37℃5%CO2培养箱中培养60h后,向各孔中加入MTS溶液40μl,继续培养2h后用酶标仪读取OD570值。刺激指数(SI)≥2时,判断为阳性;刺激指数计算公式如下:
结果:亚单位疫苗LAP致敏过的小鼠脾脏淋巴细胞经LAP、LAP抗原表位肽(LAP79-93和LAP106-120)刺激均能够发生明显的淋巴细胞增殖反应,而PBS免疫组的小鼠脾脏淋巴细胞接受上述抗原刺激时均未发生淋巴细胞增殖反应,说明多房棘球蚴亚单位疫苗LAP中抗原表位肽均保持有其免疫学特性,能够刺激机体产生针对各自Th抗原表位的细胞免疫应答(图7)。
实施例6:亚单位疫苗LAP预防效果小鼠感染多房棘球蚴研究
(1)ICR小鼠免疫
与实例4中步骤相同。
(2)ICR小鼠攻毒实验
最后一次免疫5天后,将分离好的原头蚴按1000/只接种到小鼠腹腔内,继续饲养四个月后分离小鼠腹腔内囊泡,进行称重及测量。
结果:经过多房棘球蚴亚单位疫苗LAP免疫后小鼠体内囊泡与PBS对照组相比囊泡数量和囊泡重量均明显减少。(图8~10)
以上已将本发明做一详细说明,以上所述,仅为本发明之较佳实施例而已,当不能限定本发明实施范围,即凡依本申请范围所作均等变化与修饰,皆应仍属本发明涵盖范围内。
序列表
<110> 青海大学
<120> 一种多房棘球蚴亮氨酰胺肽酶亚单位疫苗LAP及其制备方法与应用
<130> 2020.5.5
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Val Asn Pro Lys Leu Ser Glu Ser Cys Asp Leu Ile Pro Phe Pro Asn
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Claims (8)
1.一种多房棘球蚴亮氨酰胺肽酶亚单位疫苗LAP,其特征在于:其活性成分是一条多肽,其氨基酸序列如序列1所示。
2.根据权利要求1所述的多房棘球蚴亮氨酰胺肽酶亚单位疫苗LAP,其特征在于:其核苷酸序列如序列2所示。
3.一种表达载体,其特征在于:包含权利要求2所述的核苷酸序列。
4.一种转基因细胞系,其特征在于:包含权利要求2所述的核苷酸序列。
5.一种宿主菌,其特征在于:包含权利要求2所述的核苷酸序列。
6.一种多房棘球蚴亮氨酰胺肽酶亚单位疫苗LAP的制备方法,其特征在于:通过基因合成技术合成多房棘球蚴多表位肽LAP的核苷酸序列,构建含有融合基因LAP的重组表达载体pCzn1-LAP及其重组基因工程菌株Arctic Express;重组基因工程菌株Arctic Express发酵后,经Ni-IDA镍离子交换层析纯化,获得该疫苗的融合蛋白LAP。
7.权利要求1、2或6所述的多房棘球蚴亮氨酰胺肽酶亚单位疫苗LAP,其特征在于能够激发机体产生针对多房棘球蚴B细胞及T细胞的免疫应答。
8.权利要求1、2或6所述的多房棘球蚴亮氨酰胺肽酶亚单位疫苗LAP,其特征在于:在制备预防和治疗多房棘球蚴感染的药物组合中的应用。
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