CN103060284A - 一种无乳链球菌pgk亚单位重组蛋白及其制备方法 - Google Patents
一种无乳链球菌pgk亚单位重组蛋白及其制备方法 Download PDFInfo
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- CN103060284A CN103060284A CN2013100074689A CN201310007468A CN103060284A CN 103060284 A CN103060284 A CN 103060284A CN 2013100074689 A CN2013100074689 A CN 2013100074689A CN 201310007468 A CN201310007468 A CN 201310007468A CN 103060284 A CN103060284 A CN 103060284A
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Abstract
本发明提供一种无乳链球菌pgk亚单位重组蛋白及其制备方法。本发明的重组蛋白为如下1）或2）所述的蛋白：1）由序列表中序列2所示的氨基酸序列组成的蛋白质；2）将1）限定的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸且具有与序列表中序列2相同活性的由序列表中序列2衍生的蛋白质。应用本发明制备的重组蛋白进行检测时无感染、散毒危险；且本发明的制备免疫制剂的生产工艺先进、生产成本低，制备的免疫制剂性能稳定、产品附加值高，适合工厂化生产，市场应用前景广。
Description
技术领域
本发明涉及一种重组蛋白，尤其是一种无乳链球菌pgk亚单位重组蛋白。
背景技术
奶牛乳腺炎（Dairy cow mastitis）是影响世界奶牛业健康、稳定发展的最主要疫病之一。其病原有多种，但是无乳链球菌(S.agalactiae)是最主要的致病菌之一，在患乳腺炎牛乳中其检出率达到70%左右，主要引起奶牛临床型和隐性乳腺炎。世界各国科学家围绕防制奶牛乳腺炎的疫苗作出了不懈努力，最终得出的结论是传统的全菌体裂解疫苗、灭活疫苗、荚膜多糖混合疫苗对于无乳链球菌性乳腺炎均无明显预防效果，亚单位（包括单亚单位及多亚单位）免疫制剂的研发是寻求有效防控该疾病的最好途径，这是全世界相关领域认可的结论。无乳链球菌是重要的人畜共感染性致病菌，在人类主要在妇女的生殖道内居留。国外在医学领域为了预防胎儿在妊娠期或在分娩过程中婴儿的无乳链球菌感染造成败血症或肺炎，研制的传统疫苗经实践证明均无明显效果，而研制的亚单位疫苗表现良好的免疫保护作用。而奶牛无乳链球菌表面蛋白磷酸甘油酸激酶（Phosphoglycerate kinase, 简称pgk）是牛乳腺炎无乳链球菌重要的表面抗原性蛋白。因此研究和应用牛源无乳链球菌表面蛋白原核表达产物具有重要的应用价值。
发明内容
本发明提供了一种重组蛋白，其是奶牛无乳链球菌pgk亚单位重组蛋白，相应的，本发明还提供该重组蛋白的编码基因和应用。
本发明提供的一种重组蛋白，为如下1）或2）所述的蛋白：
1）由序列表中序列2所示的氨基酸序列组成的蛋白质；
2）将1）限定的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸且具有与序列表中序列2相同活性的由序列表中序列2衍生的蛋白质。
本发明的第二个目的是提供上述重组蛋白的编码基因。
优选地，所述编码基因是序列表中序列1所示的核苷酸序列。
本发明的第三个目的是提供一种无乳链球菌亚单位重组蛋白的方法，是将编码重组蛋白的基因导入到大肠杆菌中，表达获得重组蛋白。
优选地，所述大肠杆菌为BL21-DE3大肠杆菌。
本发明的第四个目的是提供所述的无乳链球菌pgk亚单位重组蛋白在检测奶牛无乳链球菌性乳腺炎感染抗体和免疫抗体中的应用。
本发明的无乳链球菌亚单位重组蛋白具有下述效果：
附图说明
图1是本发明的纯化后的无乳链球菌亚单位重组蛋白电泳图；
图2是BALB/c小鼠免疫抗体检测水平结果。
具体实施方式
实施例1
一、重组蛋白的大肠杆菌体外表达
（1）、设计扩增牛源无乳链球菌表面蛋白亚单位基因pgk基因的特异性引物；
利用引物设计软件设计了1对扩增pgk基因主要抗原优势区序列的引物。上游引物序列为5'-GGAGGATCCCTAATGGCTAAATTG-3'，下游引物序列为：5'-GACTAAGCTTTTTCAGTCAATGCTG-3'，划线部分分别为引入的限制性内切酶BamH 
和Hind 酶切位点序列。扩增片段长度984bp。见序列表1。
（2）、通过PCR扩增获得pgk基因片段；
PCR反应总体系为50μL，其中灭菌超纯化水（ddH2O） 14μL、10×PCR buffer（含Mg2+） 5μL、dNTP mixture 4μL、上游引物(10pmol/μL) 4μL、下游引物(10pmol/μL)4μL、模板DNA 4μL、Taq DNA酶( 5U/μl ) 1μL、灭菌超纯化水（ddH2O） 14μL。反应条件为预变性95℃5min；94℃1min，55℃1min，72℃1min，共30个循环；最后72℃延伸10min。将PCR产物经1%琼脂糖凝胶电泳鉴定后，根据相对分子质量Marker标示，从琼脂糖凝胶上切下含目的片段的凝胶, 按DNA快速纯化胶回收试剂盒操作步骤, 回收扩增产物。胶回收试剂盒为TAKARA产品。
（3）、对pgk基因进行克隆、测序鉴定；
将回收的目的DNA片段与pMD18-T 克隆载体于16℃连接过夜，将连接产物转化至JM109感受态细胞，用含Amp的LB琼脂平板进行筛选，提取重组质粒pgk-pMD18-T，用BamH 
和Hind 
进行单、双酶切及PCR鉴定。将转入重组质粒pgk-pMD18-T的JM109感受态细胞送至北京华大基因研究中心进行测序，测序结果用DNAStar生物软件进行分析。
（4）、将pgk基因定向亚克隆至pET-30a（+）原核表达载体；
将测序正确的重组克隆载体pgk-pMD-18与表达载体pET30a(+)用BamH
、Hind 
进行双酶切后胶回收，构建重组表达载体pgk-pET30a(+)，再将酶切及PCR鉴定正确的重组质粒pgk-pET30a(+)按常规方法转化至宿主菌大肠杆菌BL21(DE3），得到重组菌。
（5）、pgk基因在大肠杆菌的诱导表达：
将重组菌在LB液体培养基中，在37℃下培养3h，至OD600nm达到0.65左右时，加入浓度1.00 mM/L 的IPTG，在37℃下进行诱导5h,其表达量最大。其表达蛋白约占菌体总蛋白的43.2%，可溶性表达量占菌体上清总蛋白的46.5%。
二、重组蛋白的亲和层析纯化
(1)待纯化样的制备
离心收集表达细菌:将上述步骤一中得到的诱导表达蛋白于10000 rpm条件下离心10min，取沉淀,用PBS（1mol/L,pH7.4）洗涤3次，细菌沉淀被PBS溶液溶解。
裂解菌体获取上清可溶性蛋白: 加入溶菌酶至终浓度为1%(1mg/ml)，冰浴30min，然后进行超声处理，以悬液趋于透明、用移液枪吸吹时无明显凝块为度。离心取上清（即为可溶性蛋白）并加入1%的TritionX-100（聚乙二醇辛基苯基醚）及1mmol/L的DTT（二硫苏糖醇）至终浓度为1mmol/L后于-20℃保存，以备纯化。
(2)表达蛋白的亲合层析纯化流程
参照Ni-NTA His band Resin（His TrapTM FF crude，GE Heathcare 产品）操作程序进行,然后进行SDS-PAGE和Western Blot，检测蛋白纯度，如图1所示，经检测，纯度在95%以上。
Western Blot鉴定使用的一抗为兔抗牛乳腺炎无乳链球菌抗体，二抗为羊抗兔IgG HRP标记抗体。
(3)表达蛋白含量的测定
采用紫外线吸收法定量蛋白质。将待检样品适当稀释后，同时测定该蛋白质溶液在λ260nm和λ280nm处的OD值，同时用稀释液作为空白对照，按下列公式计算蛋白含量：
(1.46 × A280 - 0.74 × A260) × 稀释倍数=mg/ml蛋白含量
纯化的pgk蛋白的质量浓度为≥5.03mg/mL。
实施例2
应用本发明的重组蛋白作为检测抗原检测免疫小鼠免疫抗体的实验效果如下：
将本发明实施例1中制备的重组蛋白与弗氏完全佐剂（Freund complete adjuvant，FCA）与弗氏不完全佐剂（Freund incomplete adjuvant，FIA）按1:1等体积进行乳化，乳化用一次性无菌注射器取稀释后的抗原，另一支注射器取福氏完全佐剂或福氏不完全佐剂，排除注射器内空气后用灭菌乳胶管连接接头连接（防止压力过大时乳胶管与注射器滑脱，乳化前用细铁丝拧紧接头），首先将稀释抗原快速推入佐剂，形成油包水，然后反复快速推拉混合约10～30分钟。当乳化剂呈乳白色、放蒸馏水中水乳相不分离、5000rpm/min离心5min不分层时结束乳化，制备得到牛乳腺炎无乳链球菌的免疫疫苗。利用制备的无乳链球菌pgk重组蛋白抗原检测该免疫疫苗免疫产生的抗体情况。
本发明的pgk重组蛋白抗原检测免疫疫苗抗体效果如下：
将该免疫疫苗对BALB/c小鼠免疫三次（间隔7d）后，第一次免疫用福氏完全佐剂乳化重组抗原，剂量为1mL/只，背部皮下分3点接种；第二次用福氏不完全佐剂乳化重组抗原，剂量为1mL/只，背部皮下分3点接种；第三次免疫用非乳化重组蛋白抗原进行免疫，剂量为1mL/只，背部皮下分3点接种。第三次免疫后35天其抗体滴度达到1:4000。见图2（抗体检测水平效果）。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 内蒙古民族大学
<120> 一种无乳链球菌pgk亚单位重组蛋白及其制备方法
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<212> DNA
<213> 人工重组基因
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Glu Glu Ala Asp Lys Glu Gly Lys Ser Leu Ala Pro Val Ala Ala Asp
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Gly Ile Phe Val Asn Asp Ala Phe Gly Thr Ala His Arg Ala His Ala
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Ala Asn Ala Phe Ala Gly Tyr Thr Glu Val Arg Asp Thr Glu Gly Glu
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Claims (6)
1.一种无乳链球菌pgk亚单位重组蛋白，其特征在于：所述重组蛋白为如下1）或2）所述的蛋白：
1）由序列表中序列2所示的氨基酸序列组成的蛋白质；
2）将1）限定的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸且具有与序列表中序列2相同活性的由序列表中序列2衍生的蛋白质。
2.权利要求1所述的无乳链球菌pgk亚单位重组蛋白的编码基因。
3.根据权利要求2所述无乳链球菌pgk亚单位重组蛋白的编码基因，其特征在于：所述编码基因是序列表中序列1所示的核苷酸序列。
4.含有权利要求2或3所述编码基因的重组菌。
5.一种高效表达权利要求1所述无乳链球菌pgk亚单位重组蛋白的方法，是将编码重组蛋白的基因导入到大肠杆菌中，表达获得重组蛋白。
6.权利要求1所述的无乳链球菌pgk亚单位重组蛋白在检测奶牛乳腺炎无乳链球菌感染抗体及疫苗免疫抗体中的应用。
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Non-Patent Citations (3)
| Title |
|---|
| 《GENBANK》 20081024 Tettelin H "登录号：AAN00629.1" , * |
| TETTELIN H: ""登录号：AAN00629.1"", 《GENBANK》 * |
| 张海宝 等: "《牛乳腺炎无乳链球菌Pgk基因抗原优势区原核表达产物的抗原性分析》", 《中国动物传染病学报》 * |
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