CN104491833B - 一种治疗Ebola病毒的药物及其制备方法 - Google Patents
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Abstract
本发明公开了一种治疗Ebola病毒的药物及其制备方法，旨在提供一种制备方法简单，用于治疗埃博拉病毒的药物；其技术要点：所述的药物为特异切割Ebola病毒RNA的融合蛋白PIN‑VP30‑DD，基因序列如SEQ ID NO：1所示；制备方法为：1)PIN‑VP30‑DD原核表达载体的构建；2)PIN‑VP30‑DD蛋白的原核表达纯化；属于生物医药技术领域。
Description
技术领域
本发明公开了一种生物药物，具体地说，是一种治疗Ebola病毒的药物，本发明该公开了该药物的制备方法，属于生物医药技术领域。
背景技术
埃博拉病毒(EBOV)是引起人类和灵长类动物发生埃博拉出血热(EBHF)的烈性病毒，由此引起的出血热是当今世界上最致命的病毒性出血热，已造成10次具有规模的爆发流行。目前没有针对Ebola病毒的特效药物。Emily P等人用纳米级的脂包被的siRNA(nucleoprotein为靶基因)治疗Ebola病毒，目前这个实验还在进行之中，由于它只是抑制转录，所以很难根治。
发明内容
针对上述不足，本发明的目的在于提供一种制备方法简单，用于治疗埃博拉病毒的药物及其制备方法。
为解决上述技术问题，本发明提供的第一个技术方案是这样的：该治疗Ebola病毒的药物，所述的药物为特异切割Ebola病毒RNA的融合蛋白PIN-VP30-DD，基因序列如SEQ IDNO：1所示。
进一步的，上述的治疗Ebola病毒的药物，所述的药物通过下述步骤制备：
1)PIN-VP30-DD原核表达载体的构建
以Ebola病毒RNA为模板，进行RT-PCR，然后扩增VP30片段，并进行点突变，即突变成VP30-DD；
PCR扩增PIN，然后两个PCR片段融合，克隆到PET-16b的BamHI和NdeI位点；
2)PIN-VP30-DD蛋白的原核表达纯化
将带有重组质粒PIN-VP30-DD的大肠杆菌BL21菌种扩大培养，在12-18℃经IPTG过夜诱导，收集所得到的PIN-VP30-DD蛋白菌体，经过高压破碎后离心获得可溶性的上清，进行Ni柱亲和层析；待目的蛋白与Ni柱结合之后,先用漂洗液洗涤，在用洗脱缓冲液洗涤，在用脱盐柱将洗脱组分中的高盐缓冲液置换成含有20％甘油的PBS溶液，之后进行SDS-PAGE的检测，所得即为纯化的PIN-VP30-DD蛋白；
为制备该药物，本发明的另外一个技术方案是提供该治疗Ebola病毒药物的方法，该方法依次包括下述步骤：
1)PIN-VP30-DD原核表达载体的构建
序列合成PIN-VP30-DD，然后以PIN-VP30-DD为模板，进行PCR扩增，然后克隆到PET-16b的BamHI和NdeI位点；
2)PIN-VP30-DD蛋白的原核表达纯化
将带有重组质粒PIN-VP30-DD的大肠杆菌BL21菌种扩大培养，在12-18℃经IPTG过夜诱导，收集所得到的PIN-VP30-DD蛋白菌体，经过高压破碎后离心获得可溶性的上清，进行Ni柱亲和层析；待目的蛋白与Ni柱结合之后,先用漂洗液洗涤，在用洗脱缓冲液洗涤，在用脱盐柱将洗脱组分中的高盐缓冲液置换成含有20％甘油的PBS溶液，之后进行SDS-PAGE的检测，所得即为纯化的PIN-VP30-DD蛋白。
上述的治疗Ebola病毒药物的制备方法，所述的漂洗液由下述组分构成：20mMTris-HCl，500mM NaCl，5％甘油，60mM咪唑。
进一步的，上述的治疗Ebola病毒药物的制备方法，所述的漂洗液pH8.0。
上述的治疗Ebola病毒药物的制备方法，所述的洗脱缓冲液由下述组分构成：20mMTris-HCl，500mM NaCl，5％甘油，500mM咪唑。
进一步的，上述的治疗Ebola病毒药物的制备方法，所述的pH8.0。
进一步的，上述的治疗Ebola病毒药物的制备方法，所述的脱盐柱为PD-10脱盐柱。
与现有技术相比，本发明提供的技术方案，将一个RNA非序列特异性的切割DomainPIN与VP30-DD进行融合，构建成PIN-VP30-DD融合蛋白，该蛋白可以特异性切割Ebola病毒RNA，从而可以用来Ebola病毒感染的病毒治疗。
VP30是Ebola病毒的一个特异性RNA结合蛋白，有288个氨基酸组成，它参与这个病毒的转录开始。VP30的磷酸化，调节它与NP的结合，从而影响转录的调节。VP30在氨基酸(aa29 31 and 42 46)有两个和三个磷酸化位点，把这几个丝氨酸突变成天冬氨酸，即VP30-DD。该蛋白降低Ebola病毒mRNA的合成，但是该蛋白仍然保留了特异的RNA结合特性。
附图说明
图1是本发明提供的PIN-VP30-DD蛋白特异的切割Ebola virus病毒RNA。检测图谱。
具体实施方式
下面结合具体实施方式对本发明的权利要求做进一步的详细说明，但不构成对本发明的任何限制，任何人在本发明权利要求范围内所做的有限次的修改，仍在本发明的权利要求保护范围之内。
实施例1
本发明提供的一种治疗Ebola病毒的药物，该药物为特异切割Ebola病毒RNA的融合蛋白PIN-VP30-DD，基因序列如SEQ ID NO：1所示。
该药物由下述步骤依次制备：
1.PIN-VP30-DD原核表达载体的构建。
序列合成PIN-VP30-DDDNA融合序列，克隆到PET-16b的BamHI和NdeI位点。具体序列如SEQ ID NO：1所示：
以PIN-VP30-DD DNA融合序列为模板，用引物PET-16b_F:
TTGTTAGCAGCCGGATCCATGGAAGCTTCATATGAGAGAGGAC(SEQ ID NO：2)和引物
PET-16b_R:ATCGAAGGTCGTCATATGTTATAGCCGGATTGGCTCCTCTT(SEQ ID NO：3)进行PCR扩增，然后Infusion克隆到PET-16b的BamHI和NdeI位点。即为原核表达载体PET-16b-PIN-VP30-DD
2.PIN-VP30-DD蛋白的原核表达纯化。
将带有重组质粒PIN-VP30-DD的大肠杆菌BL21菌种扩大培养，在16℃经IPTG过夜诱导，分别收集所得到的PIN-VP30-DD蛋白菌体，经过高压破碎后离心获得可溶性的上清，进行Ni柱亲和层析。待目的蛋白与Ni柱结合之后,先用漂洗液(20mM Tris-HCl，500mM NaCl，5％(v/v)甘油，60mM咪唑，pH8.0)洗涤，之后用洗脱缓冲液(20mM Tris-HCl,500mM NaCl,5％(v/v)甘油，500mM咪唑，pH8.0)。利用PD-10脱盐柱将洗脱组分中的高盐缓冲液置换成含有20％甘油的PBS溶液，之后进行SDS-PAGE的检测。所得即为纯化的PIN-VP30-DD蛋白具体序列如SEQ ID NO：4所示。
为了更好的说明本发明的效果，下面给出PIN-VP30-DD蛋白活性的验证方法：
PIN-VP30-DD蛋白与Ebola virus病毒RNA进行孵育，经过RT-PCR检查。发现PIN-VP30-DD能够特异的切割Ebola virus病毒RNA。
具体实验如下：
PIN-VP30-DD蛋白与Ebola virus病毒RNA的切割实验。
取Ebola virus病毒感染的细胞。然后把PIN-VP30-DD克隆到真核表达载体上(慢病毒)，然后用这个病毒感染这株细胞。24小时后，用荧光RT-PCR检查Ebola virus病毒RNA，与对照相比，实验组的Ebola virus病毒RNA明显减少，甚至消失。这个实验说明，PIN-VP30-DD蛋白可以特异的切割Ebola virus病毒。
Claims (6)
1.一种治疗Ebola病毒的药物，其特征在于，所述的药物为特异切割Ebola病毒RNA的融合蛋白PIN-VP30-DD，基因序列如SEQ ID NO：1所示；
所述的药物通过下述步骤制备：
1)PIN-VP30-DD原核表达载体的构建
序列合成PIN-VP30-DD，然后以PIN-VP30-DD为模板，进行PCR扩增，然后克隆到PET-16b的BamHI和NdeI位点，即为原核表达载体PET-16b-PIN-VP30-DD；
2)PIN-VP30-DD蛋白的原核表达纯化
将带有原核表达载体PET-16b-PIN-VP30-DD的大肠杆菌BL21菌种扩大培养，在12-18℃经IPTG过夜诱导，收集所得到的PIN-VP30-DD菌体蛋白，经过高压破碎后离心获得可溶性的上清，进行Ni柱亲和层析；待目的蛋白与Ni柱结合之后,先用漂洗液洗涤，在用洗脱缓冲液洗涤，再用脱盐柱将洗脱组分中的高盐缓冲液置换成含有20％甘油的PBS溶液，之后进行SDS-PAGE的检测，所得即为纯化的PIN-VP30-DD蛋白。
2.制备权利要求1所述的治疗Ebola病毒药物的方法，其特征在于，该方法依次包括下述步骤：
所述的药物通过下述步骤制备：
1)PIN-VP30-DD原核表达载体的构建
序列合成PIN-VP30-DD，然后以PIN-VP30-DD为模板，进行PCR扩增，然后克隆到PET-16b的BamHI和NdeI位点，即为原核表达载体PET-16b-PIN-VP30-DD；
2)PIN-VP30-DD蛋白的原核表达纯化
将带有原核表达载体PET-16b-PIN-VP30-DD的大肠杆菌BL21菌种扩大培养，在12-18℃经IPTG过夜诱导，收集所得到的PIN-VP30-DD菌体蛋白，经过高压破碎后离心获得可溶性的上清，进行Ni柱亲和层析；待目的蛋白与Ni柱结合之后,先用漂洗液洗涤，在用洗脱缓冲液洗涤，再用脱盐柱将洗脱组分中的高盐缓冲液置换成含有20％甘油的PBS溶液，之后进行SDS-PAGE的检测，所得即为纯化的PIN-VP30-DD蛋白。
3.根据权利要求2所述的治疗Ebola病毒药物的制备方法，其特征在于，所述的漂洗液由下述组分构成：20mM Tris-HCl，500mM NaCl，5％甘油，60mM咪唑。
4.根据权利要求2或者3所述的治疗Ebola病毒药物的制备方法，其特征在于，所述的漂洗液pH8.0。
5.根据权利要求2所述的治疗Ebola病毒药物的制备方法，其特征在于，所述的洗脱缓冲液由下述组分构成：20mM Tris-HCl，500mM NaCl，5％甘油，500mM咪唑。
6.根据权利要求2所述的治疗Ebola病毒药物的制备方法，其特征在于，所述的脱盐柱为PD-10脱盐柱。
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