WO2024140160A1 - 人***瘤病毒hpv39 l1蛋白的表达和类病毒样颗粒及其制备方法 - Google Patents
人***瘤病毒hpv39 l1蛋白的表达和类病毒样颗粒及其制备方法 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N2710/00011—Details
- C12N2710/20011—Papillomaviridae
- C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2710/00011—Details
- C12N2710/20011—Papillomaviridae
- C12N2710/20023—Virus like particles [VLP]
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Definitions
- HPV can be roughly divided into two categories according to the benign and malignant nature of HPV-induced lesions: 1) High-risk types (such as HPV16, HPV18, HPV31, HPV33, HPV39, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, etc.): High-risk HPV is closely related to malignant tumors of various human tissues, mainly causing severe atypical hyperplasia and invasive cancer; 2) Low-risk types (such as HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV72, HPV81, etc.): Low-risk HPV can cause benign proliferative sexually transmitted diseases of epidermal cells, such as condyloma acuminatum and flat warts.
- High-risk types such as HPV16, HPV18, HPV31, HPV33, HPV39, HPV39, HPV45, HPV51, HPV52, HPV
- the inventors expressed the HPV39 L1 protein in a prokaryotic expression system based on the cost of the finished vaccine, and solved the difficulty in expressing the HPV39 L1 protein in a prokaryotic expression system. This was achieved specifically through the following improvements: the amino acid sequence of the HPV39 L1 protein was truncated, and the codons of the coding nucleotide sequence of the truncated protein were optimized to obtain an optimized coding nucleotide sequence, and finally a tag-free expression vector containing a specific SD sequence was used to achieve efficient expression and purification.
- sequence shown in SEQ ID NO.2 is as follows:
- sequence shown in SEQ ID NO.3 is as follows:
- the present invention removes the GST tag of the vector and replaces the SD sequence that can efficiently express the HPV39 type L1 protein to form a new expression vector suitable for the HPV39 L1 protein.
- the replaced SD sequence is AGGAGATATA (5' to 3').
- the present invention also provides a method for preparing HPV39 L1 VLP, comprising the following steps: adjusting the pH and salt concentration of the buffer solution in which the HPV39 L1 protein is located to enable it to self-assemble into VLP according to the step of obtaining the HPV39 L1 protein according to the method.
- the buffer includes but is not limited to Tris buffer, phosphate buffer, acetate buffer, HEPES buffer, MOPS buffer, citrate buffer, histidine buffer, borate buffer, preferably phosphate buffer;
- the pH of the buffer is between 4.75 and 5.25, and the salt concentration is between 2.0 and 4.0 M, preferably pH 4.75, pH 5.0, and pH 5.25;
- the salt concentration in the mixture is between 2.0-4.0 M, preferably 2.0 M, 2.5 M, 3.0 M, 3.5 M, 4.0 M;
- Figure 3 XA90 pKL1-HPV39-N9L1 expression electrophoresis detection results in small shake flasks.
- M marker; 1. XA90 pKL1 negative control; 2. XA90 pKL1-HPV39L1-N9-1 whole cells; 3. XA90 pKL1-HPV39L1-N9-1 supernatant; 4. XA90 pKL1-HPV39L1-N9-1 precipitate; 5. XA90 pKL1-HPV39L1-N9-2 whole cells; 6. XA90 pKL1-HPV39L1-N9-2 supernatant; 7. XA90 pKL1-HPV39L1-N9-2 precipitate; 8. XA90 pKL1 negative control.
- FIG. 4 HPV39-N9 L1 pentamer electrophoresis detection results.
- M marker; 1.
- Example 1 Construction of a tag-free expression vector containing a specific SD sequence
- PCR primers The names and sequences of PCR primers are as follows:
- Reverse primer 6p1-NdeImut-R sequence (5'to3'):
- the PCR reaction system was as follows: 10 ⁇ L of 5 ⁇ phusion HF buffer, 0.5 ⁇ L of ddH 2 O3, 2 ⁇ L of 10 mM dNTP, 1 ⁇ L of 6PNE-SDm-F, 1 ⁇ L of 6PNE-SDm-R, 5 ⁇ L of pGEX-6P-2 (diluted 20 times), and 0.5 ⁇ L of Phusion HF Enzyme.
- the PCR product was digested with DpnI and transformed into E.coli DH5 ⁇ , and monoclonal colonies were obtained after overnight culture. The monoclonal colonies were expanded and cultured, and then the vector sequences were sequenced by a professional gene sequencing company, and the clones with correct sequencing results were selected. Then the clones were expanded and plasmids were extracted from them to obtain the vectors with the NdeI restriction site successfully introduced.
- the enzyme digestion system is as follows: Cutsmart buffer 3 ⁇ l, ddH 2 O 3 ⁇ l, vector obtained in step 2 above 20 ⁇ l, NdeI 2 ⁇ l, BamHI 2 ⁇ l.
- the vector fragment was recovered by agarose gel recovery kit, and 3 ⁇ l of the obtained vector fragment was taken for electrophoresis to detect the recovery result. Then the double-digested product was filled with DNA polymerase I to fill the sticky ends.
- the reaction system was as follows: 10 ⁇ T4 DNA ligase buffer 2.5 ⁇ l, ddH 2 O1.8 ⁇ l, gel-recovered digested vector fragment 20 ⁇ l, 10 mM dNTP0.2 ⁇ l, DNA polymerase I0.5 ⁇ l, 25°C for 15 min, EDTA (final EDTA concentration was 10 mM) was added and heated at 75°C for 20 min to terminate the reaction.
- the PCR reaction system was as follows: 10 ⁇ L of 5 ⁇ phusion HF buffer, 0.5 ⁇ L of ddH 2 O3, 2 ⁇ L of 10 mM dNTP, 1 ⁇ L of 6PNE-SDm-noG-F, 1 ⁇ L of 6PNE-SDm-noG-R, 5 ⁇ L of template plasmid, and 0.5 ⁇ L of Phusion HF enzyme.
- the template DNA was transformed into E. coli DH5 ⁇ and monoclonal colonies were obtained after overnight culture. The monoclonal colonies were expanded and then sequenced by a professional gene sequencing company. The clones with correct sequencing results were selected, and then the clones were expanded and plasmids were extracted from them to obtain a vector that successfully replaced the SD sequence, removed the GST gene, and reintroduced NdeI and BamHI. At this point, the vector pKL1 was constructed.
- Example 2 Construction of an expression vector containing a codon-optimized HPV39L1 gene
- the ligation product is transformed into E.coli DH5 ⁇ for recombinant screening.
- the screened monoclonal colonies are expanded and cultured, and the plasmids are extracted, and then sequenced and verified to obtain the corresponding recombinant expression vector pKL1-HPV39L1 (the corresponding three truncated forms of the vectors include truncated forms: pKL1-HPV39L1 is truncated by 4 amino acids at the N-terminus and 29 at the C-terminus).
- a lysis buffer (20mM PB, 20mM DTT, pH8.0) at a mass volume ratio of 1:10, and then use a high-pressure homogenizer to high-pressure break the bacteria, and the breaking conditions are: 800bar, 3 times.
- the bacterial cell break liquid is then high-speed centrifuged (4°C, 12000rpm, 60min) to collect the supernatant.
- the supernatant is further precipitated by ammonium sulfate with a saturation of 30%, and the precipitate is collected by centrifugation (4°C, 12000rpm, 60min).
- HPV39-N9 L1-VLP prepared in Example 4 was investigated for long-term stability data at -70°C. The results are as follows.
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Abstract
一种人***瘤病毒HPV39型L1蛋白的表达和类病毒样颗粒及其制备方法,包括对HPV39型L1蛋白的氨基酸序列进行截短,并针对该截短后的蛋白的编码核苷酸序列进行密码子优化得到优化的编码核苷酸序列,最后配合含有特定SD序列无标签表达载体实现无标签表达纯化。通过上述改进使得在原核表达***例如大肠杆菌表达***中获得更高的蛋白表达量,且得到质量更均一的VLP。
Description
本发明涉及医药生物领域,具体涉及人***瘤病毒HPV39 L1蛋白VLP(类病毒样颗粒)的构建及表达。
人***瘤病毒(human papillomavirus,HPV)是一种无包膜的闭环双链DNA病毒,属乳多空病毒科多瘤病毒亚科,主要侵犯人体的上皮黏膜组织,进而诱发各种良性及恶性增生病变。目前已鉴定出来的不同亚型HPV超过200种,HPV感染具有明显的组织特异性,不同型别的HPV对于皮肤和黏膜的嗜向性不同,能诱发不同的***状病变,大约有30多种HPV型别与生殖道感染有关,其中有20多种与肿瘤相关。
根据HPV诱发病变的良恶性不同,HPV可大致分为两类:1)高危型(如HPV16、HPV18、HPV31、HPV33、HPV39、HPV39、HPV45、HPV51、HPV52、HPV56、HPV58、HPV59等):高危型HPV与人类多种组织恶性肿瘤密切相关,主要引起重度不典型增生和***;2)低危型(如HPV6、HPV11、HPV40、HPV42、HPV43、HPV44、HPV54、HPV72、HPV81等):低危型HPV可引起表皮细胞良性增殖性性病,如***和***等。HPV主要由病毒外壳和基因组DNA构成。基因组长约7900bp,有8个病毒蛋白编码基因。其中6个ORF编码的蛋白在病毒复制的早期表达,称为早期蛋白;2个ORF编码的蛋白在病毒复制的晚期表达,称为晚期蛋白。晚期蛋白包括主要外壳蛋白L1及次要外壳蛋白L2,并参与病毒外壳的形成。HPV病毒外壳蛋白能够进行自组装,目前在专利文献中,多采用酵母表达***或者昆虫表达***或者在哺乳动物细胞表达***里面单独表达的L1蛋白或将L1蛋白与L2蛋白共表达时均能自组装成病毒样颗粒(virus-like particle,VLP),利用外源表达体系生产的VLP免疫后能够在体内诱发产生中和抗体,获得良好的免疫保护效果。但是利用真核表达***在体内直接表达组装VLP,产生VLP的性质并不是很均一,并且真核表达***的成本很高,不利于产业化。
目前针对HPV39型在CN202110981124.2中报道,其中用的是汉逊酵母表达***产生HPV39L1蛋白,汉逊酵母表达***是真核表达***,在体内直接组装成VLP,该专利中并没有提出是否在大肠杆菌原核表达***里是否可以正常表达合格标准的蛋白,因为大肠杆菌原核表达***并不具备汉逊酵母表达***翻译后修饰等功能,所以在原核表达***里表达
HPV39 L1是有一定难度的。因此,需要研究解决在原核表达***里面表达HPV39L1蛋白困难的问题,来获得更均一的VLP以及在产业应用上有更低的成本。
发明内容
本发明人针对基于疫苗成品成本的考虑,在原核表达***里面表达HPV39 L1蛋白,并解决了在原核表达***里面表达HPV39L1蛋白困难的问题。具体通过以下改进实现:对HPV39型L1蛋白的氨基酸序列进行截短,并针对该截短后的蛋白的编码核苷酸序列进行密码子优化得到优化的编码核苷酸序列,最后配合含有特定SD序列的无标签表达载体实现高效表达和纯化。
首先,本发明将HPV39L1蛋白的氨基酸序列全长如SEQ ID NO.1所示,对其做N/C端截短处理,以期获得更好的蛋白表达率。N端截短不多于10个氨基酸,优选9个氨基酸。C端截短不多于30个氨基酸,优选29个氨基酸,具体的截短后的氨基酸见SEQ ID NO.2。N/C端截短处理后,可在无标签表达载体上表达并且获得更高质量的蛋白及VLP。
其中,SEQ ID NO.2所示序列如下:
其次,为了利用大肠杆菌***高效的表达HPV39L1蛋白,发明人根据SEQ ID NO.2所示的氨基酸序列,针对大肠杆菌***进行核苷酸序列的密码子优化。优化原则包括:a)按照大肠杆菌遗传密码使用频率表选用使用频率最高或较高的密码子;b)消除常用的限制性内切酶识别位点。通过上述原则经过优化的核苷酸序列并进行多次筛选,获得了优化后的核苷酸序列如SEQ ID NO.3所示,进一步提供含有所述编码核酸的表达盒,表达载体和重组宿主细胞。优选地,其是大肠杆菌。
其中,SEQ ID NO.3所示序列如下:
最后,本发明提供特定SD序列的无标签表达载体。对于表达载体而言,表达融合蛋白的载体pGEX的特点是在载体上具有一种26kDa的谷胱甘肽S-转移酶基因(GST),与其它的融合载体相比,它具有纯化条件温和、步骤简单、无变性剂加入,以及纯化后蛋白能最大限度保持其空间构象和免疫原性的特点;具有较好的应用价值,但载体pGEX编码的GST融合蛋白标签可能会增加药用蛋白产品的安全性隐患。对此,本发明将该载体的GST标签去除,并且替换能够高效表达HPV39型L1蛋白的SD序列,而形成新的适合HPV39 L1蛋白的表达载体。换后的SD序列为AGGAGATATA(5'to3')。
本发明还提供一种制备HPV39型L1VLP的方法,包括如下步骤:根据所述的方法得到的HPV39型L1蛋白的步骤,调节其所在缓冲液的pH和盐浓度,使其自组装形成VLP。
优选地,所述缓冲液包括但不限于Tris缓冲液,磷酸盐缓冲液,醋酸缓冲液,HEPES缓冲液,MOPS缓冲液,枸橼酸缓冲液、组氨酸缓冲液,硼酸缓冲液,优选磷酸盐缓冲液;
缓冲液的pH在4.75-5.25,盐浓度在2.0-4.0M之间,优选pH4.75,pH5.0,pH5.25;其
中的盐浓度在2.0-4.0M之间,优选2.0M,2.5M,3.0M,3.5M,4.0M;
本发明通过上述改进使得在原核例如大肠杆菌表达***中可以获得更高的蛋白表达量,且得到质量更均一的VLP。
图1 XA90 pKL1-HPV39L1小摇瓶表达电泳检测结果。其中,M:marker;1.XA90pKL1阴性对照;2.XA90 pKL1-HPV39L1-1全菌;3.XA90 pKL1-HPV39L1-1上清;4.XA90 pKL1-HPV39L1-1沉淀;5.HPV18L1;6.XA90 pKL1-HPV39L1-2全菌;7.XA90 pKL1-HPV39L1-2上清;8.XA90 pKL1-HPV39L1-2沉淀。
图2 XA90 pKL1-HPV39-FL L1小摇瓶表达电泳检测结果。其中,M:marker;1.XA90pKL1阴性对照;2.XA90 pKL1-HPV39-FL L1全菌;3.XA90 pKL1-HPV39-FL L1上清;4.XA90 pKL1-HPV39-FL L1沉淀;5.XA90pKL1阴性对照;6.XA90 pKL1-HPV39-FL L1-2全菌;7.XA90 pKL1-HPV39-FL L1-2上清;8.XA90 pKL1-HPV39-FL L1-2沉淀。
图3 XA90 pKL1-HPV39-N9L1小摇瓶表达电泳检测结果。其中,M:marker;1.XA90pKL1阴性对照;2.XA90 pKL1-HPV39L1-N9-1全菌;3.XA90 pKL1-HPV39L1-N9-1上清;4.XA90 pKL1-HPV39L1-N9-1沉淀;5.XA90 pKL1-HPV39L1-N9-2全菌;6.XA90 pKL1-HPV39L1-N9-2上清;7.XA90 pKL1-HPV39L1-N9-2沉淀;8.XA90pKL1阴性对照。
图4 HPV39-N9 L1五聚体电泳检测结果。其中,M:marker;1.HPV39-N9 L1五聚体。
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作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。
实施例一:含有特定SD序列的无标签表达载体的构建
1.通过突变PCR,在pGEX-6P-2质粒中引入NdeI酶切位点:
PCR引物名称及序列如下:
正向引物:6p1-NdeImut-F(5'to3'):
反向引物:6p1-NdeImut-R序列(5'to3'):
PCR反应体系如下:5×phusion HF缓冲液10μL,ddH2O30.5μL,10mM dNTP 2μL,6PNE-SDm-F 1μL,6PNE-SDm-R 1μL,pGEX-6P-2(稀释20倍)5μL,Phusion HF Enzyme 0.5μL。
PCR反应程序设置:95℃3min;95℃1min,55℃1min,72℃10min;循环20次;72℃15min。
PCR产物用DpnI消化后转化到E.coliDH5α中,过夜培养后获得单克隆菌落。将单克隆菌落进行扩大培养,之后由专业的基因测序公司对其中的载体序列进行测序,选出测序结果正确的克隆,然后将克隆扩繁并从中提取质粒,获得成功引入NdeI酶切位点的载体。
2.设计替换SD序列的突变PCR引物,然后通过PCR方法替换原载体的SD序列
引物信息如下:
6PNE-SDm-F(5'to3'):CAATTTCACACAGGAGATATACATATGTCCCCTATACTAGG
6PNE-SDm-R(5'to3'):GTATAGGGGACATATGTATATCTCCTGTGTGAAATTGTTATCC
PCR反应体系如下:5×phusion HF缓冲液10μL,ddH2O30.5μL,10mM dNTP 2μL,6PNE-SDm-F 1μL,6PNE-SDm-R 1μL,前述步骤获得的质粒5μL,Phusion HF酶0.5μL。
PCR反应程序设置:95℃3min;95℃1min,55℃1min,72℃10min;循环20次;72℃15min。
PCR产物用DpnI消化模板DNA后,转化到E.coliDH5α中,过夜培养后获得单克隆菌落。将单克隆菌落进行扩大培养,之后由专业的基因测序公司对其中的载体序列进行测序,选出测序结果正确的克隆,然后将克隆扩繁并从中提取质粒,获得成功替换SD序列的载体。替换后的SD序列为AGGAGATATA(5'to3')。
3.载体进行NdeI和BamHI双酶切去除GST基因
酶切体系如下:Cutsmart缓冲液3μl,ddH2O3μl,上述步骤2获得的载体20μl,NdeI 2μl,BamHI 2μl。
37℃酶切2h;0.8%琼脂糖凝胶电泳,120V,1h;切胶获得去除GST基因后的载体片段对应电泳条带,4℃保存。
用琼脂糖凝胶回收试剂盒回收载体片段,所得载体片段取3μl电泳检测回收结果。然后将双酶切产物用DNA聚合酶I补齐粘性末端,反应体系如下:10×T4DNA连接酶缓冲液2.5μl,ddH2O1.8μl,胶回收的酶切载体片段20μl,10mM dNTP0.2μl,DNA聚合酶I0.5μl,25℃反应15min,加入EDTA(EDTA终浓度为10mM)并且75℃加热20min终止反应。
将酶切后并末端补齐的载体进行重新连接环化,连接体系如下:T4DNA连接酶缓冲液
2μl,线性平末端载体片段16μl,T4DNA连接酶2μl。16℃连接4h。
连接产物消化后转化到E.coliDH5α中,过夜培养后获得单克隆菌落。将单克隆菌落进行扩大培养,之后由专业的基因测序公司对其中的载体序列进行测序,选出测序结果正确的克隆,然后将克隆扩繁并从中提取质粒,获得成功替换SD序列并去除GST基因的载体。
4.PCR扩增质粒,重新引入NdeI和BamHI的酶切位点
PCR引物如下:
6PNE-SDm-noG-F(5'to3'):CAGGAGATATACATATGGGATCCCCGGAATTCCCG
6PNE-SDm-noG-R(5'to3'):GAATTCCGGGGATCCCATATGTATATCTCCTGTGTG
PCR反应体系如下:5×phusion HF缓冲液10μL,ddH2O30.5μL,10mM dNTP 2μL,6PNE-SDm-noG-F 1μL,6PNE-SDm-noG-R 1μL,模板质粒5μL,Phusion HF酶0.5μL。
PCR反应程序设置:95℃3min;95℃1min,55℃1min,72℃10min;循环20次;72℃15min。
PCR产物用DpnI消化模板DNA后,转化到E.coliDH5α中,过夜培养后获得单克隆菌落。将单克隆菌落进行扩大培养,之后由专业的基因测序公司对其中的载体序列进行测序,选出测序结果正确的克隆,然后将克隆扩繁并从中提取质粒,获得成功替换SD序列、去除GST基因,并重新引入NdeI和BamHI的载体。至此,载体pKL1构建完毕。
实施例二:含密码子优化的HPV39L1基因的表达载体的构建
人***瘤病毒39型外壳蛋白L1(HPV39L1,其全长氨基酸序列如SEQ ID NO.1所示)进行了三种截短形式,分别是N端截短4个氨基酸,C端截短29个氨基酸;N端全长,C端截短29个氨基酸;以及N端截短9个氨基酸,C端截短29个氨基酸(其氨基酸序列如SEQ ID NO.2所示,优化密码子后的编码核苷酸序列如SEQ ID NO.3所示),上述截短后的编码核苷酸序列通过人工合成。
先PCR扩增HPV39L1的DNA片段,将含有NdeI和Xho1酶切位点的L1基因PCR片段以及重组载体pKL1分别进行NdeI和Xho1双酶切,之后利用T4DNA连接酶将回收的基因片段与含有对应粘性末端的pKL1载体进行连接反应,16℃10~15h。连接体系如下:pKL1载体片段6μl,前述的HPV39L1基因片段2μl,T4DNA连接酶1μl,T4DNA连接酶缓冲液1μl。连接反应后转化连接产物到E.coli DH5α中进行重组子的筛选。将筛选的单克隆菌落进行扩大培养并进行质粒的提取,之后进行测序验证,得到相应的重组表达载体pKL1-HPV39L1(相应前述三种截短形式的载体包括截短形式分别为:pKL1-HPV39L1是N端截短4个氨基酸,C端截短29个
氨基酸;pKL1-HPV39-FL L1是N端全长,C端截短29个氨基酸,pKL1-HPV39-N9 L1是N端截短9个氨基酸,C端截短29个氨基酸)。
实施例三:HPV39 L1蛋白的表达
将实施例二测序结果正确的三种重组载体pKL1-HPV39L1转化大肠杆菌XA90宿主细胞,并作为表达重组蛋白质的工程菌进行HPV L1蛋白的表达。0.05%的接种量接种至LB培养基(Amp+)中,于37℃,220rpm培养16h进行活化。取活化后菌液按0.5%的接种量接种至2YT培养基中,于30℃,220rpm培养7h后添加终浓度为0.2mM的IPTG,于30℃,220rpm诱导培养16h后结束发酵,离心收集菌体用于表达量检测以及纯化实验。从SDS-PAGE的结果来看(图1和图2),表达载体pKL1-HPV39L1(N端截短4个氨基酸,C端截短29个氨基酸),以及pKL1-HPV39-FL L1(N端全长,C端截短29个氨基酸)的两种载体,小摇瓶表达检测结果显示其不能有效表达目的蛋白。从图3中可见,只有XA90pKL1-HPV39-N9 L1(N端截短9个氨基酸,C端截短29个氨基酸)能够有效表达目的蛋白,虽然有一部分目的蛋白形成了包涵体(在沉淀中),但破菌上清里亦有较多的目的蛋白。可见,通过对N端和C端做不同优化,得到不同的实验结果,N端截短九个氨基酸更能够带来意想不到的技术效果,目的蛋白单位菌体表达量约为0.2mg/g湿菌体。
实施例四:HPV39L1蛋白纯化及VLP组装
取适量XA90 pKL1-HPV39-N9 L1菌体按质量体积比1:10的比例用破菌缓冲液(20mM PB,20mM DTT,pH8.0)充分重悬,然后用高压均质机对菌体进行高压破碎,破碎条件为:800bar,3次。菌体破碎液接着进行高速离心(4℃,12000rpm,60min)收集上清。上清进一步通过饱和度为30%的硫酸铵沉淀,离心(4℃,12000rpm,60min)收集沉淀,沉淀按质量体积比1:10的比例用复溶缓冲液(20mM PB,20mM DTT,pH8.0)充分复溶后再次离心(4℃,12000rpm,60min)收集上清获得粗纯液。粗纯液先上样进行Superdex200分子筛层析,分子筛缓冲液(20mM PB,20mM DTT,pH8.0),根据L1目的蛋白的出峰位置收集其所在组分。接着将分子筛收集样品上样进行Source15Q阴离子交换层析(SQ低盐缓冲液:5mM PB,10mM DTT,pH8.0,SQ高盐缓冲液:5mM PB,1M NaCl,10mM DTT,pH8.0),通过0-20%高盐缓冲液,10个柱体积线性洗脱收集L1目的蛋白所在组分,该组分即为纯化后L1蛋白。通过动态光散射(DLS)法测定L1五聚体的质量。五聚体的SDS-PAGE电泳图见图4。可见在目的蛋白区域形成五聚体单一、纯度很高,并且表达量高。最后调节L1蛋白所在缓冲液的pH和盐浓度至其自组装形成VLP,至此VLP的制备完成。最后通过DLS测定
VLP的质量。
表1 HPV39-N9 L1蛋白组装前后DLS检测结果
纯化实验结果显示的HPV39-N9 L1的五聚体状态良好(PdI≤0.1),也能有效组装形成状态良好的VLP(45nm≤粒径大小≤75nm,PdI≤0.1)
实施例五:蛋白长期稳定性实验
取实施例四制备的HPV39-N9 L1-VLP,在-70℃条件下,进行长期稳定性数据的考察,考察结果如下。
“-”表示无此项规定或者该项未进行试验。
可以看出,经过9个月的长期考察,HPV39-N9 L1-VLP抗原蛋白的外观性状,pH值,VLP平均粒径及分散系数,纯度以及体外效价等,都没有明显变化,抗原蛋白相当稳定。
最后应说明的是:以上所述仅为本发明的优先实施例而已,并不用来限制本发明,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种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内。
Claims (10)
- 一种截短的HPV39型L1蛋白,其是在野生型HPV39型L1蛋白的基础上,在其N端截短不多于10个氨基酸,优选9个氨基酸,且在其C端截短不多于30个氨基酸,优选29个氨基酸;优选地,截短的HPV39型L1蛋白的氨基酸序列如SEQ ID NO.2所示。
- 编码如权利要求1所述的截短的HPV39型L1蛋白的核酸;优选地,其经过密码子优化的核酸;更优选地,其核苷酸序列如SEQ ID NO.3所示。
- 含有SD序列和编码如权利要求2所述的截短的HPV39型L1蛋白的核苷酸序列的核酸,优选地,所述SD序列的核苷酸序列为5`-AGGAGATATA-3`。
- 含有如权利要求2或3所述的编码核酸的表达盒或表达载体。
- 如权利要求4所述的表达盒或表达载体,其特征在于,其是原核表达载体,更优选地是在载体pGEX基础上去除了GST标签序列,并且整合所述SD序列的截短的HPV39型L1蛋白的核酸分子而得到。
- 含有如权利要求2或3所述的编码核酸的表达盒或表达载体的重组宿主细胞。
- 如权利要求6所述的重组宿主细胞,其特征在于,其是原核细胞,优选为大肠杆菌。
- 一种表达如权利要求1截短的HPV39型L1蛋白的方法,其特征在于,培养如权利要求6或7所述的重组宿主细胞以产生HPV39型L1蛋白,任选地,包括纯化步骤,优选地所述纯化步骤为:取所述的重组宿主细胞的菌体用破菌缓冲液充分重悬,然后用高压均质机对菌体进行高压破碎,离心收集上清;上清进一步通过硫酸铵沉淀,硫酸铵的最终饱和度为30%,沉淀复溶后再次离心收集上清获得粗纯液;粗纯液先上样进行Superdex200分子筛层析,根据L1目的蛋白的出峰位置收集其所在组分;接着将分子筛收集样品上样进行Source15Q阴离子交换层析,通过NaCl线性洗脱收集L1目的蛋白所在组分,得到HPV39型L1蛋白。
- 一种表达如权利要求1截短的HPV39型L1蛋白的方法,其特征在于,包括如下步骤:根据如权利要求8所述的方法得到的HPV39型L1蛋白的步骤,调节其所在缓冲液的pH和盐浓度,使其自组装形成VLP。
- 如权利要求9所述的方法,其特征在于,所述缓冲液包括但不限于Tris缓冲液,磷酸盐缓冲液,醋酸缓冲液,HEPES缓冲液,MOPS缓冲液,枸橼酸缓冲液、组氨酸缓冲液,硼酸缓冲液,优选磷酸盐缓冲液;缓冲液的pH在4.75-5.25,盐浓度在2.0-4.0M之间,优选pH4.75,pH5.0,pH5.25;其 中的盐浓度在2.0-4.0M之间,优选2.0M,2.5M,3.0M,3.5M,4.0M;任选地,还包括纯化所得HPV39L1五聚体的步骤。
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CN102229660A (zh) * | 2011-05-25 | 2011-11-02 | 厦门大学 | 截短的人***瘤病毒33型l1蛋白 |
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CN110551183A (zh) * | 2018-06-04 | 2019-12-10 | 厦门大学 | 一种人***瘤病毒39型l1蛋白的突变体 |
CN116041444A (zh) * | 2022-12-28 | 2023-05-02 | 北京康乐卫士生物技术股份有限公司 | 人***瘤病毒hpv39 l1蛋白的表达和类病毒样颗粒及其制备方法 |
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