WO2017107060A1 - 一种新型病毒样颗粒表达载体、其构建方法及应用 - Google Patents

一种新型病毒样颗粒表达载体、其构建方法及应用 Download PDF

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WO2017107060A1
WO2017107060A1 PCT/CN2015/098298 CN2015098298W WO2017107060A1 WO 2017107060 A1 WO2017107060 A1 WO 2017107060A1 CN 2015098298 W CN2015098298 W CN 2015098298W WO 2017107060 A1 WO2017107060 A1 WO 2017107060A1
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plasmid
virus
expression vector
gene
particle
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邓俊花
吴绍强
林祥梅
张永宁
吕继洲
冯春燕
王彩霞
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中国检验检疫科学研究院
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Priority to US15/329,881 priority Critical patent/US20180127837A1/en
Priority to PCT/CN2015/098298 priority patent/WO2017107060A1/zh
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Definitions

  • the pyrimidine interacts with the envelope protein. 2
  • the number of packaging sites is different.
  • the same expression vector, while introducing the exogenous fragment, increases the packaging site, which greatly increases the length of the packaging segment and the packaging efficiency.
  • the promoter of the expression vector is different, and the ability to transcribe RNA is also different.
  • Qiuying et al. (2006) used pSE380 as a vector to express VLPs containing exogenous chimeric RNA containing SARS-CoV2 and HCV 1200 bp by deleting the unnecessary multiple cloning sites on the pAR-1 vector using pSE380 as a vector. .
  • a novel virus-like particle expression vector pTMSCA2C of the present invention is based on the plasmid pTrcHis-MS2, and firstly uses the gene mutation technique to construct the plasmid pTrcHis-MS2 on the MS2 phage 19mer packaging site gene sequence.
  • the 5th base of the T mutation is C, and the plasmid pTMSC is obtained; then the plasmid pTMSC is compiled.
  • plasmid pTMSCA Preparation of plasmid pTMSCA: The mutated base was introduced into the PCR primer, and the plasmid pTMSC was used as a template, and Primer1 was used as a primer to carry out PCR amplification to obtain PCR product I, and the purified amplification product was recovered; pTMSC plasmid was used for NcoI and PmaCI. Double digestion, and then using the In-Fusion technology to link the PCR product I with the digested pTMSC plasmid, transforming the recipient bacteria, screening and identification, to obtain the plasmid pTMSCA;
  • PrimerA, PrimerB and Primer1 have the following primer sequences:
  • PrimerA-F 5'-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3’
  • PrimerA-R 5'-TGGGTGATCCTCATGTTTGAATGGCCGGCGTC-3';
  • PrimerB-F 5'-GCCATTCAAACATGAGGATCACCCATGTCGAAG-3’
  • PrimerB-R 5'-GTTCGGGCCCAAGCTTCGAATTCCC-3';
  • Primer1-F 5'-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3’
  • Primer1-R 5'-CCACCTGCCGGCCACGTGTTTTGATC-3'.
  • the primers for identification in steps 1) and 2) are Primer-U1: 5'-GACAATTAATCATCCGGCTCG-3' and Primer-L1: 5'-GATCTTCGTTTAGGGCAAGGTAG-3'.
  • the present invention also provides a virus-like particle comprising a novel virus-like particle expression vector pTMSCA2C and a RNA transcript carrying the foreign gene.
  • the invention also provides a method for preparing a virus-like particle, which comprises cloning a foreign gene fragment into the sequence of the MS2 phage coat protein gene coding sequence in tandem in the novel virus-like particle expression vector pTMSCA2C, and inserting a terminator downstream thereof, after transcription A foreign gene RNA transcript with a bacteriophage operon RNA sequence is obtained, which induces expression of the phage coat protein and assembles into a protein coat, and encapsulates the foreign gene RNA transcript carried in the protein coat to obtain virus-like particles.
  • the invention also provides the use of the novel virus-like particle expression vector or the virus-like particle in preparing a quality control product for detecting pathogenic microorganisms.
  • the invention further provides a control for the detection of pathogenic microorganisms prepared from the virus-like particles.
  • the present invention also provides the use of the virus-like particles in the detection of animal pathogenic microorganisms, mainly as a reference substance, and is applied to various animal pathogenic microorganism nucleic acid detection technologies.
  • the AB-loop hairpin structure increases the protein purification tag, which affects the coat protein packaging efficiency and reduces the production of virus-like particles.
  • the novel virus-like particle expression vector contains a nucleotide sequence encoding a double CP protein structure. Under the function of ensuring the AB-loop hairpin structure to increase the protein purification label, it will not reduce the efficiency of the foreign protein packaged by the coat protein. At the same time, the mutation of the start codon of the mature enzyme protein also effectively increased the yield of the finally obtained virus-like particles.
  • Lane 1 is a comparative effect of a recombinant strain containing the plasmid pTMSCA and a recombinant strain containing the plasmid pTrcHis-MS2 to induce a cleavage supernatant of the expression product for nucleic acid electrophoresis in Example 1.
  • Lane 1 is a recombinant plasmid-inducing product cleavage supernatant (5 ⁇ L) containing plasmid pTMSCA
  • Lane 2 is a recombinant plasmid-inducing expression product cleavage supernatant (5 ⁇ L) containing plasmid pTrcHis-MS2
  • M is DL2000 DNA Marker.
  • 2 is a comparative effect of the nucleic acid electrophoresis of the cleavage supernatant of the expression product after the recombinant plasmid containing the plasmid pTMSCA and the recombinant plasmid containing the plasmid pTrcHis-MS2 are packaged with different lengths of the exogenous fragment in the first embodiment of the present invention.
  • FIG. 3 is a comparison effect of the novel virus-like particle expression vector pTMSCA2C, the recombinant plasmid containing the plasmid pTMSCA and the vector pTrcMS in packaging the cleavage supernatant of the expression product for nucleic acid electrophoresis in Example 2 of the present invention.
  • lane 1 pTMSCA2C-SBV virus-like particle (Coomassie blue staining)
  • M is Spectra TM Multicolor Low Range Protein Ladder.
  • Fig. 5 is a fluorescence diagram showing the presence or absence of DNA residue by real-time PCR after purification of the virus-like particle pTMSCA2C-SBV in Example 3 of the present invention.
  • Fig. 6 is an electron microscopic observation chart (100000 ⁇ JEM1400) of the virus-like particle pTMSCA2C-SBV in Example 3 of the present invention.
  • PrimerA-R 5'-TGGGTGATCCTCATGTTTGAATGGCCGGCGTC-3';
  • the PCR amplification procedure was: denaturation at 98 ° C for 10 s, annealing at 55 ° C for 10 s, extension at 72 ° C for 30 s, 30 cycles, and extension at 72 ° C for 10 min to recover and purify the amplified product.
  • the 2pTrcHis-MS2 plasmid was digested with XhoI and HindIII, and the system was as follows: XhoI/HindIII10U, 5 ⁇ L of 10 ⁇ digestion buffer, 1 ⁇ g of plasmid template, and ddH 2 O to 10 ⁇ L. The reaction was carried out at 37 ° C for 2 h. The digested products were separately subjected to gel recovery and purified and designated as pTrcHis-MS2 (X/H).
  • Primer1-R 5'-CCACCTGCCGGCCACGTGTTTTGATC-3’
  • 9pTMSCA and pTrcHis-MS2 cleavage supernatant contrast effect recombinant strain pTMSCA
  • the cells were induced by IPTG (final concentration of 1 mol/L) with pTrcHis-MS2 for 16 h, 5000 rpm, and centrifuged for 10 min, respectively, and the cells were collected.
  • the product precipitate was added to a 1 ⁇ TE buffer (pH 8.0) 20 ⁇ L/mL cell pellet, vortexed, and lysozyme (25 mg/mL) 1 ⁇ L/mL was added, and the mixture was digested at 37 ° C for 30 min. Normal temperature, 10000 rpm, centrifuge for 10 min.
  • the pTMSCA and pTrcHis-MS2 supernatants were subjected to nucleic acid electrophoresis to compare the yield of the expressed product (Fig. 1).
  • the first CP gene sequence is the gene coding sequence of the wild phage MS2 coat protein.
  • the second CP gene sequence is inserted into the tandem structure of the wild phage MS2 coat protein gene coding sequence before the coding sequence of the coat protein gene of the pTrcMS vector (SEQ ID NO: 2) to obtain a novel virus-like particle expression vector pTMSCA2C.
  • the specific construction method is as follows:
  • plasmids pTMSCA2C, pTMSC-AP and pTrcMS were constructed according to step 10 of Example 1, respectively, to construct pTMSCA2C-D (D > 1800 bp), pTMSCA-D (D > 1800 bp) and pTrcMS-A (A ⁇ 500 bp).
  • Three recombinant bacteria were operated according to step 9) of Example 1, and the yield of the expressed product was compared (Fig. 3).
  • the pGEM-T-SBV plasmid and the pTMSCA2C plasmid prepared in Example 2 were separately digested with KpnI and HindIII, purified by gel, ligated, and sequenced to construct a recombinant plasmid pTMSCA2C-SBV.
  • the recovered virus-like particles were extracted and subjected to real-time PCR and RT-PCR to verify the purity of the solution.
  • the primers for PCR identification were Primer-SBV-F: 5'-TCAGATTGTCATGCCCCTTGC-3'; Primer-SBV-R: 5'-TTCGGCCCCAGGTGCAAATC-3'. Probe: 5'-FAM-TTAAGGGATGCACCTGGGCCGATGGT-3'. (SEQ ID NO: 12-14)
  • the purified virus-like particle solution was first stained with 1% uranyl acetate, then naturally dried, and finally observed by transmission electron microscopy.
  • the purified SBV virus-like particles were used as positive control products, and SBV nucleic acid was detected in clinically collected sheep serum samples.
  • the purified SBV virus-like particles were detected by SDS-PAGE. The results are shown in Figure 4.
  • the target protein is located between 26kDa and 48kDa, which is consistent with the MS2 phage coat protein twice the size (27.4kDa).
  • the purity verification results show that the PCR identification is not expanded. Increasing the curve, RT-PCR identification showed a standard S amplification curve indicating that the solution contained the virus-like particles of the Schmallenberg target gene and was free of DNA contamination (Fig. 5); electron microscopy showed that the diameter was approximately 26nm polygonal particles (Fig.

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Abstract

提供一种新型病毒样颗粒表达载体pTMSCA2C，首先采用基因突变技术将质粒pTrcHis-MS2上MS2噬菌体19mer包装位点基因序列中的第5位碱基T突变为C，得到质粒pTMSC；然后将质粒pTMSC上编码MS2噬菌体成熟酶蛋白的起始密码子对应的氨基酸由缬氨酸突变成甲硫氨酸，得到质粒pTMSCA；最后采用基因克隆技术，将野生型MS2噬菌体外壳蛋白基因编码序列去掉终止子后，与来自假病毒载体pTrcMS的含组氨酸蛋白标签的MS2噬菌体外壳蛋白的基因编码序列进行串联，并将串联后的基因序列连接到质粒pTMSCA中构建得到表达载体pTMSCA2C。

Description

一种新型病毒样颗粒表达载体、其构建方法及应用 技术领域
本发明涉及基因工程领域，具体地说，涉及一种新型病毒样颗粒表达载体、其构建方法及应用。
背景技术
二十世纪末，Pasloske等人提出了一种新的RNA质控品制备技术，即装甲RNA(Armored RNA)技术。该技术的原理是将包含有大肠杆菌MS2噬菌体的外壳蛋白基因的序列及外源基因克隆到表达载体中，这一载体能够将外源基因转录成RNA，并利用载体上MS2的外壳蛋白编码基因组装的外壳蛋白将其装配成球状RNA病毒结构的RNA-蛋白质复合体，称之为装甲RNA病毒样颗粒。
传统Armored RNA制备技术中病毒样颗粒(Virus-Like-Particles,VLPs)表达载体包装外源RNA片段通常超过500bp时，包装效率显著降低。设计一种病毒样颗粒表达载体，提高包装外源RNA长度的能力，进而在RNA病原微生物临床检测的质量控制和标准化上具有更为广泛的应用价值。例如，将不同病原微生物的靶基因构建在一起形成嵌合体，并包装于外壳蛋白内形成VLPs，这种VLPs就可以同时对多个病原微生物进行检测质控，既节省了成本，又减化了操作程序；另外，就某个特定的病原微生物检测而言，不同厂家生产的RT-PCR试剂盒的扩增靶片段可能有所不同，如果将这些不同的目的片段构建到同一条RNA链上，并包装入MS2外壳蛋白内形成VLPs，则不同的实验室之间就可以对结果进行直接的比较。
由于病毒样颗粒表达载体会随着包装位点的亲和力和数量的不同，所能包装的外源片段长度不同。①包装位点的类型不同。野生型的MS2噬菌体19mers，外源片段超过500bp，包装效率就会逐步降低，表达出来的病毒样颗粒的数量就会大大减少，纯化的病毒样颗粒就会 相应减少；通过基因突变手段对MS219mers的部分位点进行改造，载体包装外源片段的长度就会大大提高。MS2RNA的19mer的茎环结构(发夹结构)中仅4个核苷酸的位置对外壳蛋白的识别特别重要，在保证茎碱基配对情况下，4、7和10位的腺嘌呤以及5位的嘧啶与包膜蛋白相互作用。②包装位点的数量不同。同一种表达载体，在引入外源片段的同时，增加包装位点，会大大提高包装片段的长度和包装效率。同时，表达载体的启动子不同，转录RNA的能力也不同。Qiuying等(2006)以pSE380为载体，应用单质粒表达系统，通过删除pAR-1载体上的不必要的多克隆位点，表达了内含SARS-CoV2和HCV 1200bp的外源嵌合体RNA的VLPs。魏玉香等(2008)以pET-28(b)为承载体，增加MS2包装位点，表达了内含H5N1、HCV及SARS-CoV 1891bp的外源嵌合体RNA的VLPs。但是，上述研究仅是在一定程度上解决了包装外源片段长度的问题，并没有改变病毒样颗粒繁琐的制备工艺。
ZL201110445022.5公开了一种假病毒载体pTrcMS，通过将MS2噬菌体中外壳蛋白的第15-16氨基酸之间加入6His纯化标签，利用柱纯化蛋白的方式获得病毒样颗粒，大大简化了病毒样颗粒制备的繁琐操作过程，同时提高了病毒样颗粒纯化的质量。但是，以pTrcMS载体为基础制备的病毒样颗粒产量会明显降低，同样制约了病毒样颗粒的推广应用。
发明内容
本发明的目的是提供一种新型病毒样颗粒表达载体pTMSCA2C。
本发明的另一目的是提供新型病毒样颗粒表达载体pTMSCA2C的构建方法和应用。
为了实现本发明目的，本发明的一种新型病毒样颗粒表达载体pTMSCA2C，其是以质粒pTrcHis-MS2为出发载体，首先采用基因突变技术将质粒pTrcHis-MS2上MS2噬菌体19mer包装位点基因序列中的第5位碱基T突变为C，得到质粒pTMSC；然后将质粒pTMSC上编 码MS2噬菌体成熟酶蛋白的起始密码子对应的氨基酸由缬氨酸突变成甲硫氨酸，得到质粒pTMSCA；最后采用基因克隆技术，将野生型MS2噬菌体外壳蛋白基因编码序列去掉终止子后，与来自假病毒载体pTrcMS的含组氨酸蛋白标签(6His蛋白纯化标签)的MS2噬菌体外壳蛋白的基因编码序列进行串联，并将串联后的基因序列(串联后的基因序列编码双CP蛋白结构)连接到质粒pTMSCA中构建得到的。其中，质粒pTrcHis-MS2(ZL201110445022.5)的核苷酸序列如SEQ ID NO:1所示，假病毒载体pTrcMS的核苷酸序列如SEQ ID NO:2所示。
本发明的新型病毒样颗粒表达载体pTMSCA2C的核苷酸序列如SEQ ID NO:3所示。
本发明还提供所述表达载体的构建方法，包括以下步骤：
1)质粒pTMSC的制备：在PCR引物中引入突变的碱基，以质粒pTrcHis-MS2为模板，分别以PrimerA、PrimerB为引物，进行PCR扩增，分别得到PCR产物A、B，回收纯化扩增产物；pTrcHis-MS2质粒用XhoⅠ和HindⅢ双酶切，然后利用In-Fusion技术将PCR产物A、B与酶切后的pTrcHis-MS2质粒连接，转化受体菌，经筛选、鉴定，得到质粒pTMSC；
2)质粒pTMSCA的制备：在PCR引物中引入突变的碱基，以质粒pTMSC为模板，以Primer1为引物，进行PCR扩增，得到PCR产物I，回收纯化扩增产物；pTMSC质粒用NcoI和PmaCI双酶切，然后利用In-Fusion技术将PCR产物I与酶切后的pTMSC质粒连接，转化受体菌，经筛选、鉴定，得到质粒pTMSCA；
3)表达载体pTMSCA2C的构建：将野生型MS2噬菌体外壳蛋白基因编码序列去掉终止子后，与来自假病毒载体pTrcMS的含组氨酸蛋白标签的MS2噬菌体外壳蛋白的基因编码序列进行串联，并将串联后的基因序列插入到质粒pTMSCA的XhoⅠ和HindⅢ酶切位点之间，即构建得到表达载体pTMSCA2C。
其中，PrimerA、PrimerB和Primer1的引物序列如下：
PrimerA-F:5’-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3’
PrimerA-R:5’-TGGGTGATCCTCATGTTTGAATGGCCGGCGTC-3’；
PrimerB-F:5’-GCCATTCAAACATGAGGATCACCCATGTCGAAG-3’
PrimerB-R:5’-GTTCGGGCCCAAGCTTCGAATTCCC-3’；
Primer1-F:5’-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3’
Primer1-R:5’-CCACCTGCCGGCCACGTGTTTTGATC-3’。
前述的方法，步骤1)和2)中鉴定用引物为Primer-U1:5’-GACAATTAATCATCCGGCTCG-3’和Primer-L1:5’-GATCTTCGTTTAGGGCAAGGTAG-3’。
本发明还提供一种病毒样颗粒，所述病毒样颗粒含有新型病毒样颗粒表达载体pTMSCA2C及其携带外源基因RNA转录本。
本发明还提供一种病毒样颗粒的制备方法，将外源基因片段克隆于所述新型病毒样颗粒表达载体pTMSCA2C中串联的MS2噬菌体外壳蛋白基因编码序列下游，在其下游插入终止子，转录后得到带有噬菌体操纵子RNA序列的外源基因RNA转录本，诱导表达噬菌体外壳蛋白并组装成蛋白外壳，并将携带的外源基因RNA转录本包裹到蛋白外壳内，得到病毒样颗粒。
本发明还提供所述新型病毒样颗粒表达载体或所述病毒样颗粒在制备检测病原微生物质控品中的应用。
本发明进一步提供由所述病毒样颗粒制备的检测病原微生物的质控品。
此外，本发明还提供所述病毒样颗粒在动物病原微生物检测中的应用，主要作为参比物质，应用于各类动物病原微生物核酸检测技术。
利用本发明的表达载体pTMSCA2C制备病毒样颗粒，在提高病毒样颗粒产量和纯度的同时，可以极大降低病毒样颗粒制备的工作量。本发明制备的病毒样颗粒，具有以下优点：
(一)增加外源基因的包装容量。由于噬菌体MS2基因组中19mer的茎环结构碱基发生改变，大大提高了包装位点与外壳蛋白的亲和力，长片段的外源基因能顺利包装到外壳蛋白内，形成VLPs。
(二)易纯化。由于外壳蛋白AB-loop发卡结构处添加了能任意扭转的6His蛋白纯化标签，并且还有双外壳蛋白的基因结构，大大提高了外壳蛋白的裸露面积，增强了外壳蛋白与蛋白纯化系统的亲和力。经诱导表达之后，蛋白纯化标签裸露在外壳蛋白的表面，利用6His蛋白纯化方法捕获病毒样颗粒，可以大大减少病毒样颗粒制备的繁琐操作过程，同时提高病毒样颗粒的纯度。
(三)获得极高产量。在单个外壳蛋白结构状态下，AB-loop发卡结构增加蛋白纯化标签，会影响外壳蛋白包装效率，降低病毒样颗粒的产量；新型病毒样颗粒表达载体中含有编码双CP蛋白结构的核苷酸序列，在保证AB-loop发卡结构增加蛋白纯化标签的功能下，不会降低外壳蛋白包装外源基因的效率。同时，成熟酶蛋白起始密码子突变后，也有效提高了最终获得病毒样颗粒的产量。
附图说明
图1为本发明实施例1中含有质粒pTMSCA的重组菌与含有质粒pTrcHis-MS2的重组菌诱导表达产物裂解上清进行核酸电泳的对比效果。其中，泳道1为含有质粒pTMSCA的重组菌诱导表达产物裂解上清(5μL)；泳道2为含有质粒pTrcHis-MS2的重组菌诱导表达产物裂解上清(5μL)，M为DL2000 DNA Marker。
图2为本发明实施例1中含有质粒pTMSCA的重组菌与含有质粒pTrcHis-MS2的重组菌包装不同长度的外源片段后表达产物裂解上清进行核酸电泳的对比效果。其中，M为DL2000 DNA Marker，泳道1为含有质粒pTrcHis-MS2的重组菌包装外源基因片段A(A<500bp)后的表达产物裂解上清(5μL)；泳道2为含有质粒pTrcHis-MS2的重组菌包装外源基因片段B(1000bp>B>500bp)后的表达产物裂解上清 (5μL)；泳道3为含有质粒pTMSCA的重组菌包装外源基因片段C(1500bp>C>1000bp)后的表达产物裂解上清(5μL)；泳道4为含有质粒pTMSCA的重组菌包装外源基因片段D(D>1800bp)后的表达产物裂解上清(5μL)。
图3为本发明实施例2中新型病毒样颗粒表达载体pTMSCA2C、含有质粒pTMSCA的重组菌与载体pTrcMS包装不同长度的外源片段后表达产物裂解上清进行核酸电泳的对比效果。其中，M为1kb DNA Marker，泳道1为pTMSCA包装外源基因片段D(D>1800bp)后的表达产物裂解上清(5μL)；泳道2为pTrcMS包装外源基因片段A(A<500bp)后的表达产物裂解上清(5μL)；泳道3为pTMSCA2C包装外源基因片段D(D>1800bp)后的表达产物裂解上清(5μL)。
图4为本发明实施例3中病毒样颗粒pTMSCA2C-SBV纯化后SDS-PAGE电泳检测结果。其中，泳道1为pTMSCA2C-SBV病毒样颗粒(考马斯亮蓝染色)，M为SpectraTM Multicolor Low Range Protein Ladder。
图5为本发明实施例3中病毒样颗粒pTMSCA2C-SBV纯化后real-time PCR检测有无DNA残留的荧光图。
图6为本发明实施例3中病毒样颗粒pTMSCA2C-SBV的电镜观察图(100000×JEM1400)。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(SambrookJ&RussellDW,Molecularcloning:alaboratorymanual,2001)，或按照制造厂商说明书建议的条件。
实施例1含有质粒pTMSCA的重组菌的构建
以pTrcHis-MS2质粒(SEQ ID NO:1)为模板，采用In-Fusion技术先将MS2噬菌体19mer包装位点基因序列中的5位碱基T突变为C，得 到pTMSC；然后将编码MS2噬菌体成熟酶蛋白的起始密码子对应氨基酸由缬氨酸突变成甲硫氨酸，得到质粒pTMSCA。
具体构建方法如下：
①在PCR引物中引入突变的碱基，以质粒pTrcHis-MS2为模板，分别以PrimerA/PrimerB为引物对，用
Figure PCTCN2015098298-appb-000001
STAR HS DNA Polymerase分别进行PCR扩增，扩增产物命名为PCR产物A、B。其中，PCR扩增引物为(SEQ ID NO:4-7)：
PrimerA-F:5’-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3’
PrimerA-R:5’-TGGGTGATCCTCATGTTTGAATGGCCGGCGTC-3’；
PrimerB-F:5’-GCCATTCAAACATGAGGATCACCCATGTCGAAG-3’
PrimerB-R:5’-GTTCGGGCCCAAGCTTCGAATTCCC-3’。
50μL PCR扩增的反应体系为：
Figure PCTCN2015098298-appb-000002
STAR Buffer(Mg2+plus)10μL,
Figure PCTCN2015098298-appb-000003
STAR HS DNA聚合酶1.3U，dNTP Mixture(各2.5mM)4μL,PrimerA-F/R(20pmol/μL)各0.5μL，PrimerB-F/R(20pmol/μL)各0.5μL,pTrcHis-MS2DNA模板10ng，补ddH2O至50μL。PCR扩增程序为：98℃变性10s，55℃退火10s，72℃延伸30s，30个循环，72℃延伸10min，回收纯化扩增产物。
②pTrcHis-MS2质粒采用XhoⅠ和HindⅢ双酶切，体系如下：XhoⅠ/HindⅢ10U，10×酶切缓冲液5μL，质粒模板1μg，补ddH2O至10μL。37℃反应2h。分别将酶切产物进行胶回收纯化,命名为pTrcHis-MS2(X/H)。
③使用
Figure PCTCN2015098298-appb-000004
HD Cloning Kit(Clontech Code No.639648)，将PCR产物A、B和pTrcHis-MS2(X/H)连接，反应体系及条件如下：PCR产物A/B各200ng,pTrcHis-MS2(X/H)100ng,5×In-Fusion HD Enzyme Premix 2μL，补ddH2O至10μL。50℃反应15min。
④取上述In-Fusion产物2.5μL热转化至E.coli Competent Cell JM109，37℃过夜培养，挑取阳性菌落进行测序鉴定，鉴定用引物为 (Primer-U1:5’-GACAATTAATCATCCGGCTCG-3’，Primer-L1:5’-GATCTTCGTTTAGGGCAAGGTAG-3’，SEQ ID NO:10-11)，将测序正确的质粒命名为pTMSC。
⑤在PCR引物中引入突变的碱基，以质粒pTMSC为模板，以Primer1为引物对，用
Figure PCTCN2015098298-appb-000005
STAR HS DNA Polymerase分别进行PCR扩增，扩增产物命名为PCR产物I。其中，PCR扩增引物为(SEQ ID NO:8-9)：
Primer1-F:5’-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3’
Primer1-R:5’-CCACCTGCCGGCCACGTGTTTTGATC-3’
50μL PCR扩增的反应体系为：
Figure PCTCN2015098298-appb-000006
STAR Buffer(Mg2+plus)10μL，
Figure PCTCN2015098298-appb-000007
STAR HS DNA聚合酶1.3U，dNTP Mixture(各2.5mM)4μL，Primer1-F/R(10pmol/μL)各0.5μL，pTMSC DNA模板10ng,补ddH2O至50μL。PCR扩增程序为：98℃变性10s，55℃退火10s，72℃延伸30s，30个循环，72℃延伸10min，回收纯化扩增产物。
⑥pTMSC质粒采用NcoI和PmaCI双酶切，体系如下：NcoI/PmaCI 10U，10×酶切缓冲液5μL，质粒模板1μg，补ddH2O至10μL。37℃反应2h。分别将酶切产物进行胶回收纯化，命名为pTMSC(N/P)。
⑦使用
Figure PCTCN2015098298-appb-000008
HD Cloning Kit(Clontech Code No.639648)，将PCR产物Ⅰ和pTMSC(N/P)连接，反应体系及条件如下：PCR产物I200ng,pTMSC(N/P)100ng,5×In-Fusion HD Enzyme Premix 2μL，补ddH2O至10μL。50℃反应15min。
⑧取上述In-Fusion产物2.5μL热转化至E.coli Competent Cell JM109，37℃过夜培养，挑取阳性菌落进行测序鉴定，鉴定用引物为(Primer-U1:5’-GACAATTAATCATCCGGCTCG-3’，Primer-L1:5’-GATCTTCGTTTAGGGCAAGGTAG-3’)，将测序正确的质粒命名为pTMSCA。
⑨pTMSCA与pTrcHis-MS2裂解上清对比效果：重组菌pTMSCA 与pTrcHis-MS2分别采用IPTG(终浓度为1mol/L)诱导表达16h，5000rpm，离心10min，收集菌体。产物沉淀加入1×TE缓冲液(pH8.0)20μL/mL菌体沉淀，涡旋混匀，加入溶菌酶(25mg/mL)1μL/mL，37℃消化30min。常温，10000rpm，离心10min。pTMSCA与pTrcHis-MS2上清进行核酸电泳，比较表达产物的产量(图1)。
⑩pTMSCA与pTrcHis-MS2包装不同长度的外源片段后表达产物裂解上清的对比效果：质粒pTrcHis-MS2借助KpnI和HindIII两个酶切位点分别与不同长度的外源基因片段A(A<500bp)和B(500bp<B<1000bp)进行连接、转化、测序，构建重组质粒pTrcHis-MS2-A和pTrcHis-MS2-B；pTMSCA参照pTrcHis-MS2-A和pTrcHis-MS2-B构建方法分别构建了重组质粒pTMSCA-C(1000bp<C<1500bp)和pTMSCA-D(D>1800bp)。四种重组菌参照步骤⑨进行操作，比较表达产物的产量(图2)。
实施例2新型病毒样颗粒表达载体pTMSCA2C的构建
以pTMSCA质粒为模板，采用酶切插入方法，在XhoI/HindIII酶切位点之间插入编码MS2噬菌体外壳蛋白的双CP串联合成基因：第一CP基因序列为野生噬菌体MS2外壳蛋白的基因编码序列并且删除终止密码子，第二CP基因序列为在pTrcMS载体(SEQ ID NO:2)外壳蛋白基因编码序列之前插入野生噬菌体MS2外壳蛋白基因编码序列构成串联结构，得到新型病毒样颗粒表达载体pTMSCA2C。具体构建方法如下：
①以pTMSCA质粒为起始载体，将MS2噬菌体外壳蛋白对应的基因编码序列按以下列模式进行改造：第一CP基因序列为野生MS2噬菌体外壳蛋白的基因编码序列并且删除终止密码子，第二CP基因序列为pTrcMS载体外壳蛋白基因编码序列，合成的2CP序列连入pUC57载体中，由华大基因生物有限公司进行合成，将重组载体命名为pUC57-2CP。
②pUC57-2CP和pTMSCA质粒分别采用XhoⅠ和HindⅢ双酶切，体系如下：XhoⅠ/HindⅢ10U，10×酶切缓冲液5μL，质粒模板1μg，补ddH2O至10μL。37℃反应2h。分别将酶切产物进行胶回收纯化，命名为pUC57-2CP(X/H)和pTMSCA(X/H)。
③使用DNA连接试剂盒(Takara,Code No.6022)，将pUC57-2CP(X/H)和pTMSCA(X/H)连接，反应体系及条件如下：pUC57-2CP(X/H)/pTMSCA(X/H)各100ng,溶液I 5μL,补ddH2O至10μL。16℃连接过夜。
④取上述连接产物2.5μL热转化至E.coli Competent Cell JM109，37℃过夜培养，挑取阳性菌落进行测序鉴定，鉴定用引物为(Primer-U1:5’-GACAATTAATCATCCGGCTCG-3’，Primer-L1:5’-GATCTTCGTTTAGGGCAAGGTAG-3’)，将测序正确的质粒命名为pTMSCA2C。
⑤pTMSCA2C、pTMSCA与pTrcMS(ZL201110445022.5)包装不同长度的外源片段后表达产物裂解上清的对比效果：质粒pTMSCA2C、pTMSC-AP与pTrcMS参照实施例1步骤⑩，分别构建了pTMSCA2C-D(D>1800bp)、pTMSCA-D(D>1800bp)和pTrcMS-A(A<500bp)。三种重组菌参照实施例1步骤9)进行操作，比较表达产物的产量(图3)。
实施例3施马伦贝格(SB)病毒样颗粒的制备
1、构建pTMSCA2C-SBV质粒
pGEM-T-SBV质粒与实施例2制备的pTMSCA2C质粒分别进行KpnI和HindIII双酶切，胶回收纯化，连接，测序，构建重组质粒pTMSCA2C-SBV。
2、pTMSCA2C-SBV病毒样颗粒的制备
①表达及裂解：具体方法参照实施例1步骤⑨进行。
②pTMSCA2C-SBV上清，采用Ni Sepharose 6Fast Flow纯化系统(GE)回收产物，收集到的滤液即为纯化后的病毒样颗粒 pTMSCA2C-SBV，产物进行蛋白电泳SDS-PAGE鉴定。
③回收的病毒样颗粒抽提RNA，分别进行real-time PCR和RT-PCR，以验证该溶液的纯度。PCR鉴定引物分别为Primer-SBV-F:5’-TCAGATTGTCATGCCCCTTGC-3’；Primer-SBV-R:5’-TTCGGCCCCAGGTGCAAATC-3’。探针:5’-FAM-TTAAGGGATGCACCTGGGCCGATGGT-3’。(SEQ ID NO:12-14)
3、pTMSCA2C-SBV病毒样颗粒形态学鉴定
纯化后的病毒样颗粒溶液先进行1％醋酸双氧铀染色，然后自然干燥，最后透射电子显微镜进行形态观察。
4、pTMSCA2C-SBV病毒样颗粒临床应用
以纯化的SBV病毒样颗粒为阳性质控品，对临床采集绵羊血清样本进行SBV核酸检测。
纯化的SBV病毒样颗粒经SDS-PAGE电泳检测，结果见图4，目的蛋白位于26kDa与48kDa之间，与MS2噬菌体外壳蛋白2倍大小(27.4kDa)相符；纯度验证结果表明：PCR鉴定无扩增曲线，RT-PCR鉴定显示标准S扩增曲线，表明该溶液中含有施马伦贝格靶基因的病毒样颗粒，并且无DNA污染(图5)；经电镜观察表明，看到直径大约为26nm的多边形颗粒(图6)，即诱导表达的病毒样颗粒；临床应用实验表明：经荧光定量检测，SBV病毒样颗粒阳性质控核酸检测为标准S曲线，临床血清样本核酸检测为阴性，与样本血清学检测结果一致。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明公开了一种新型病毒样颗粒表达载体及其构建方法。利用本发明的表达载体制备病毒样颗粒，在提高病毒样颗粒产量和纯度的同时，可以极大降低病毒样颗粒制备的工作量。
参考文献
[1]Pasloske B L,et al.Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards.J Clin Microbiol,1998,36(12):3590-3594；Walker Peach C R,et al.Ribonuclease resistant RNA controls(Armored RNA)for reverse transcription-PCR,branched DNA,and genotyping assays for hepatitis C virus.Clin Chem,1999,45(12):2079-2085
[2]Talbot SJ,Goodman S,Bates SR,et al.Use of synthetic oligoribonucleotides to probe RNA-protein interactions in the MS2translational operator complex.Nucleic Acids RES,1990,18(12):3521-3528
[3]Qiuying H,Yangjian C,Qiwei G,et al.Preparation of a chimeric Armored RNA as a versatile calibrator for multiple virus assays.Clin Chem,2006,52(7):1446-1448
[4]Yuxiang Wei,Changmei Yang,Baojun Wei,et al.RNase-Resistant virus-like particles containing long chimeric RNA sequences produced by two-plasmid coexpression system.Journal of clinical microbiology,2008,46(5):1734-1740
Figure PCTCN2015098298-appb-000009
Figure PCTCN2015098298-appb-000010
Figure PCTCN2015098298-appb-000011
Figure PCTCN2015098298-appb-000012
Figure PCTCN2015098298-appb-000013
Figure PCTCN2015098298-appb-000014
Figure PCTCN2015098298-appb-000015

Claims (8)

  1. 一种新型病毒样颗粒表达载体pTMSCA2C，其特征在于，其是以质粒pTrcHis-MS2为出发载体，首先采用基因突变技术将质粒pTrcHis-MS2上MS2噬菌体19mer包装位点基因序列中的第5位碱基T突变为C，得到质粒pTMSC；然后将质粒pTMSC上编码MS2噬菌体成熟酶蛋白的起始密码子对应的氨基酸由缬氨酸突变成甲硫氨酸，得到质粒pTMSCA；最后采用基因克隆技术，将野生型MS2噬菌体外壳蛋白基因编码序列去掉终止子后，与来自假病毒载体pTrcMS的含组氨酸蛋白标签的MS2噬菌体外壳蛋白的基因编码序列进行串联，并将串联后的基因序列连接到质粒pTMSCA中构建得到的；
    其中，质粒pTrcHis-MS2的核苷酸序列如SEQ ID NO:1所示，假病毒载体pTrcMS的核苷酸序列如SEQ ID NO:2所示。
  2. 根据权利要求1所述的表达载体，其特征在于，表达载体pTMSCA2C的核苷酸序列如SEQ ID NO:3所示。
  3. 权利要求1或2所述表达载体的构建方法，其特征在于，包括以下步骤：
    1)质粒pTMSC的制备：在PCR引物中引入突变的碱基，以质粒pTrcHis-MS2为模板，分别以PrimerA、PrimerB为引物，进行PCR扩增，分别得到PCR产物A、B，回收纯化扩增产物；pTrcHis-MS2质粒用XhoⅠ和HindⅢ双酶切，然后利用In-Fusion技术将PCR产物A、B与酶切后的pTrcHis-MS2质粒连接，转化受体菌，经筛选、鉴定，得到质粒pTMSC；
    2)质粒pTMSCA的制备：在PCR引物中引入突变的碱基，以质粒pTMSC为模板，以Primer1为引物，进行PCR扩增，得到PCR产物I，回收纯化扩增产物；pTMSC质粒用NcoI和PmaCI双酶切，然后利用In-Fusion技术将PCR产物I与酶切后的pTMSC质粒连接，转化受体菌，经筛选、鉴定，得到质粒pTMSCA；
    3)表达载体pTMSCA2C的构建：将野生型MS2噬菌体外壳蛋白基因编码序列去掉终止子后，与来自假病毒载体pTrcMS的含组氨酸蛋白标签的MS2噬菌体外壳蛋白的基因编码序列进行串联，并将串联后的基因序列插入到质粒pTMSCA的XhoⅠ和HindⅢ酶切位点之间，即构建得到表达载体pTMSCA2C；
    其中，PrimerA、PrimerB和Primer1的引物序列如下：
    PrimerA-F:5’-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3’
    PrimerA-R:5’-TGGGTGATCCTCATGTTTGAATGGCCGGCGTC-3’；
    PrimerB-F:5’-GCCATTCAAACATGAGGATCACCCATGTCGAAG-3’
    PrimerB-R:5’-GTTCGGGCCCAAGCTTCGAATTCCC-3’；
    Primer1-F:5’-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3’
    Primer1-R:5’-CCACCTGCCGGCCACGTGTTTTGATC-3’。
  4. 根据权利要求3所述的方法，其特征在于，步骤1)和2)中鉴定用引物为Primer-U1:5’-GACAATTAATCATCCGGCTCG-3’和Primer-L1:5’-GATCTTCGTTTAGGGCAAGGTAG-3’。
  5. 一种病毒样颗粒，其特征在于，含有权利要求1或2所述新型病毒样颗粒表达载体pTMSCA2C及其携带外源基因RNA转录本。
  6. 一种病毒样颗粒的制备方法，其特征在于，将外源基因片段克隆于权利要求1或2所述新型病毒样颗粒表达载体pTMSCA2C中串联的MS2噬菌体外壳蛋白基因编码序列下游，在其下游插入终止子，转录后得到带有噬菌体操纵子RNA序列的外源基因RNA转录本，诱导表达噬菌体外壳蛋白并组装成蛋白外壳，并将携带的外源基因RNA转录本包裹到蛋白外壳内，得到病毒样颗粒。
  7. 权利要求1或2所述新型病毒样颗粒表达载体或权利要求5所述病毒样颗粒在制备检测病原微生物质控品中的应用。
  8. 由权利要求5所述病毒样颗粒制备的检测病原微生物的质控品。
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