WO2017214940A1 - 特异促进Cplx2基因高表达的慢病毒表达载体及其应用 - Google Patents

特异促进Cplx2基因高表达的慢病毒表达载体及其应用 Download PDF

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WO2017214940A1
WO2017214940A1 PCT/CN2016/086064 CN2016086064W WO2017214940A1 WO 2017214940 A1 WO2017214940 A1 WO 2017214940A1 CN 2016086064 W CN2016086064 W CN 2016086064W WO 2017214940 A1 WO2017214940 A1 WO 2017214940A1
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cplx2
gene
sequence
expression vector
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毛侃琅
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毛侃琅
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    • A—HUMAN NECESSITIES
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    • A61P25/00—Drugs for disorders of the nervous system
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors

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  • the invention belongs to the technical field of genetic engineering, and particularly relates to a lentiviral expression vector which specifically promotes high expression of Cplx2 gene, and a construction method and application thereof.
  • AD Alzheimer's disease
  • This disease is common in the elderly. It is a degenerative degenerative disease of the central nervous system with progressive cognitive impairment and memory impairment. The clinical manifestations are the deterioration of cognitive and memory functions, the progressive decline of daily living ability, and Various neuropsychiatric symptoms and behavioral disorders. More onset in the old age, latent onset, slow and irreversible, clinically based on intelligent damage.
  • Complexin protein also known as synaphin protein
  • SNARE N-methylmaleimide-sensitive factor attachment protein receptor
  • Binding is an important neurotransmitter release regulatory protein in presynaptic structures.
  • Complexin has been found to have four subtypes, of which Complexin 2 (Cplx2) is abundantly expressed in the brain. The results of several studies showed that the expression of Cplx2 was significantly down-regulated in the hippocampus of AD patients, indicating that abnormal expression of Cplx2 may be involved in the spatial memory impairment of Alzheimer's disease. Therefore, the study of the role of Cplx2 in Alzheimer's disease is urgent.
  • there is a lack of a lentiviral expression vector that specifically promotes high expression of the Cplx2 gene which greatly limits the progress of the study.
  • the inventors conducted extensive research on the selection of vectors, recombinant construction methods, and the like, and found that the cDNA sequence of Cplx2 gene containing the EcoR I cleavage site and the Spe I restriction site was included.
  • the lentiviral expression vector which specifically promotes high expression of the Cplx2 gene can be successfully constructed by inserting into the multiple cloning site of the pLVX-IRES-Puro expression vector, thereby completing the present invention.
  • the present invention provides a lentiviral expression vector which specifically promotes high expression of the Cplx2 gene, including the basic sequence of the pLVX-IRES-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence and the Cplx2 gene cDNA sequence;
  • the multiple cloning site comprises an EcoR I cleavage site and an Spe I cleavage site
  • the Cplx2 gene cDNA sequence comprises an EcoR I cleavage site, a Cplx2 gene coding sequence and a Spe I cleavage site, the Cplx2 gene
  • the cDNA sequence is inserted positively into the multiple cloning site sequence.
  • the lentiviral expression vector constructed by inserting the cDNA sequence of Cplx2 gene into the pLVX-IRES-Puro expression vector has the advantages of high transfection efficiency and low dosage, and can stably, efficiently and stably increase the expression of Cplx2 gene.
  • the utility model can be used as a powerful tool for preparing and researching and developing Cplx2 gene expression for diseases such as Alzheimer's disease.
  • the Cplx2 gene coding sequence is obtained by PCR amplification
  • the PCR primer comprises an upstream primer and a downstream primer
  • the sequence of the upstream primer is: 5'-GCGAATTCATGGACTTCGTCATGAAGCAG-3', ie SEQ ID NO: 1.
  • the sequence of the downstream primer is: 5'-GACTAGTTTACTTCTTGAACATGTCCTGC-3', ie SEQ ID NO: 2.
  • the Cplx2 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the Cplx2 gene, which reduces the cost of sequence synthesis and has a lower cost.
  • the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the Cplx2 gene, and comprises the following steps:
  • A) Cplx2 gene primer design According to the Cplx2 gene coding sequence, using Oligo 7 analysis, select 5'-GCGAATTCATGGACTTCGTCATGAAGCAG-3', ie SEQ ID NO: 1 as the upstream primer, select 5'-GACTAGTTTACTTCTTGAACATGTCCTGC-3', ie SEQ ID NO : 2 as a downstream primer, then synthesizing the upstream primer and the downstream primer; the upstream primer and the downstream primer are free of primer dimer, and the annealing temperature difference is small;
  • PCR amplification is performed using the upstream primer and the downstream primer to obtain a large number of Cplx2 gene coding sequences, and then the sequence is subjected to an A-tail reaction, and then ligated to the pGM- by T4 DNA ligase.
  • the ligation product was obtained on the T vector, and the ligation product was transformed into competent E. coli DH5 ⁇ , uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid was picked and subjected to preliminary identification by PCR.
  • Preliminary identification results indicate that the cDNA sequence of Cplx2 gene is inserted
  • the successful bacterial liquid was sequenced and identified; the correct E. coli was identified by liquid LB medium, and the pGM-T vector carrying the cDNA sequence of Cplx2 gene was extracted, and the restriction enzymes EcoR I and Spe I were used. Enzyme digestion, electrophoresis, and gel-cutting recover a fragment of about 500 bp, which is the cDNA sequence of Cplx2 gene;
  • a lentiviral vector that specifically promotes high expression of Cplx2 gene the plasmid pLVX-IRES-Puro was extracted, digested with restriction endonuclease EcoR I enzyme and Spe I enzyme, electrophoresis, gel-removed vector, and T4DNA Ligation of the Cplx2 gene cDNA sequence into the pLVX-IRES-Puro expression vector to obtain a ligation product, the ligation product was transformed into competent E. coli DH5 ⁇ , uniformly coated onto an ampicillin-containing LB medium plate, and picked The positive monoclonal colonies were cultured and preserved, and the PCR was initially identified. The preliminary identification results indicated that the Cplx2 gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification;
  • the invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of Cplx2 gene, and after successful identification, the packaged virus is transferred into RGC5 cells, and the cells are selected by puromycin, and then real-time fluorescent quantitative PCR and Western Blot technique are respectively used. The change of Cplx2 gene expression was verified from mRNA and protein levels. The experimental results showed that the Cplx2 gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IR ES-Puro expression vector, and the Cplx2 gene expression was promoted specifically, continuously, efficiently and stably. .
  • the present invention also provides a use of a lentiviral expression vector which specifically promotes high expression of a Cplx2 gene for the preparation of a medicament for treating a disease associated with abnormal expression of a Cplx2 gene.
  • the lentiviral expression vector which specifically promotes high expression of Cplx2 gene provided by the invention has the advantages of high transfection efficiency, low dosage, specific, sustained, high efficiency and stable promotion of high expression of Cplx2 gene, and can be used as a powerful tool for Cplx2.
  • the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the Cplx2 gene, which has a good operation effect and reduces the cost of sequence synthesis. , the cost is lower.
  • Figure 1 is a plasmid map of the pLVX-IRES-Puro expression vector.
  • Fig. 2 is a schematic diagram showing the results of quantitative PCR detection after puromycin screening of cells.
  • RGC5 cells were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, 293FT cells were purchased from Thermo Fisher, Premix PrimeSTAR HS enzyme, lentiviral expression vector pLVX-IRES-Puro, virus packaging auxiliary kit, and Lenti-X GoStix kit were purchased from Takara.
  • the company, RNeasy Mini Kit was purchased from QIAGEN, pGM-T was purchased from Tiangen, and Endo-Free Plasmid Mini Kit II was purchased from Omega Bio-tek.
  • Example 2 Construction of a lentiviral vector that specifically promotes high expression of the Cplx2 gene
  • the coding sequence of the Cplx2 gene was amplified by Premix PrimeSTAR HS enzyme, and after electrophoresis recovery, it was subjected to an A-tail reaction, and then ligated to the pGM-T vector by T4 DNA ligase to obtain a ligation product ( Cplx2-T vector), the ligation product was transformed into competent E.
  • the bacteria with the correct sequencing result were cultured in liquid LB medium for 14 hours, and then the recombinant T vector containing the Cplx2 gene sequence was extracted, and the pLVX-IRES-Puro vector was firstly treated with EcoR I enzyme and Spe I enzyme. After digestion, electrophoresis was carried out, and the product was recovered by T4 DNA ligase, and then transformed into competent E. coli DH5 ⁇ , uniformly coated on an ampicillin-containing LB medium plate, cultured at 37 ° C for 12 h, and a negative control group 1 was set at the same time.
  • the competent cells were uniformly coated on the ampicillin-free plate, the negative control group 2 (the competent cells were uniformly coated on the plate containing 100 ⁇ g/ml ampicillin), and the positive control group 1 (the double digestion was performed)
  • the ligation product of the empty carrier was uniformly coated on a plate containing 100 ⁇ g/ml ampicillin, and the positive control group 2 (the empty carrier was uniformly coated on a plate containing 100 ⁇ g/mL ampicillin).
  • the experimental group grew a single colony, and the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, and the positive control group 2 did not grow colonies.
  • the previously prepared recombinant plasmid broth was taken out, and 20 ⁇ L was inoculated into 15 ml of LB medium (containing 100 ⁇ g/ml ampicillin), cultured at 37 ° C, 300 rpm for 16 h, and the recombinant plasmid pLVX-Cplx2 was extracted with Endo-Free Plasmid Mini Kit II. The purity and concentration were measured, and the results are shown in Table 2.
  • 293FT cells were cultured, and cells with good growth status were inoculated into six wells, 100000 cells per well, and the extracted recombinant plasmid pLVX-Cplx2 was transfected into 293FT cells with lentiviral packaging auxiliary kit. After 48 hours, the cells were collected. The supernatant medium of the virus was filtered with a 0.45 ⁇ m sieve for infection of RGC5 cells, and the Lenti-X GoStix kit was used to detect virus titers of 5000000 to 50000000 IFU.
  • Example 5 Fluorescence quantitative PCR was used to detect the expression level of Cplx2 gene.
  • Primers were designed using primer design software Oligo 7.0 based on the GAPDH and Cplx2 gene mRNA sequences.
  • RGC5 cells, pLVX empty vector control RGC5 cell group, and pLVX-Cplx2 high expressing cells were inoculated into 6-well plates, respectively.
  • the total RNA of each group was extracted with RNeasy Mini Kit, and the mRNA was reverse-transcribed into cDNA using PrimeScrip RT reagent Kit. Reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C , hehe. After the end of the reverse transcription, the cDNA was diluted with 90 ⁇ L of RNase Free dH20 and stored at -20 ° C for later detection.
  • the lentiviral expression vector provided by the invention specifically promoting high expression of the Cplx2 gene has high transfection efficiency.
  • the utility model has the advantages of low dosage, specific, sustained, high-efficiency and stable promotion of high expression of Cplx2 gene, and can be used as a powerful tool for drug research and development related to Cplx2; the invention also provides a slow promotion of high expression of Cplx2 gene.
  • the method for constructing the virus expression vector has good operation effect, reduces the cost of sequence synthesis, and has low cost.

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Abstract

一种特异促进Cplx2基因高表达的慢病毒表达载体，包括pLVX-IRES-puro表达载体的基本序列、抗性基因序列、多克隆位点序列、启动子序列和Cplx2基因cDNA序列；所述多克隆位点包括EcoR I酶切位点和Spe I酶切位点，所述Cplx2基因cDNA序列包括EcoR I酶切位点、Cplx2基因编码序列和Spe I酶切位点，所述Cplx2基因cDNA序列正向插入所述多克隆位点序列中。慢病毒表达载体具有转染效率高、用量少、可特异、持续、高效、稳定地表达Cplx2基因的优点，可作为有力工具应用于与Cplx2相关的药物研究与开发。

Description

特异促进Cplx2基因高表达的慢病毒表达载体及其应用 技术领域
本发明属于基因工程技术领域，尤其涉及一种特异促进Cplx2基因高表达的慢病毒表达载体及其构建方法与应用。
背景技术
阿尔茨海默病(Alzheimer’s disease，AD)，是引起老年性痴呆的最常见原因。此疾病常见于老年人，是一种进行性认知障碍和记忆能力损害为主的中枢神经系统退行性变性疾病，临床表现为认知和记忆功能不断恶化，日常生活能力进行性减退，并有各种神经精神症状和行为障碍。多起病于老年期，潜隐起病，病程缓慢且不可逆，临床上以智能损害为主。
技术问题
Complexin蛋白，又称synaphin蛋白，可以与N-已基马来酰亚胺敏感因子结合蛋白受体(soluble Nethylmaleimide-sensitive factor attachment proteinreceptor，SNARE)复合物中的突触融合蛋白(synataxin)选择性的结合，是突触前结构中重要的神经递质释放调节蛋白。目前为止发现Complexin有四个亚型，其中Complexin 2(Cplx2)主要在脑中丰富表达。多个研究的结果显示在AD患者大脑海马中Cplx2表达量显著下调，说明Cplx2表达异常可能参与了阿尔兹海默症空间记忆的损伤，因此对Cplx2在阿尔兹海默症中作用的研究刻不容缓，但现有技术中却缺乏特异促进Cplx2基因高表达的慢病毒表达载体，极大地限制了研究的进行。
问题的解决方案
技术解决方案
为解决现有技术中存在的问题，发明人在载体的选择、重组构建方法等方面进行了大量的探索研究，发现将包含EcoR I酶切位点和Spe I酶切位点的Cplx2基因cDNA序列插入pLVX-IRES-Puro表达载体的多克隆位点中可成功构建特异促进Cplx2基因高表达的慢病毒表达载体，从而完成本发明。
本发明提供一种特异促进Cplx2基因高表达的慢病毒表达载体，包括pLVX-IRES-puro表达载体的基本序列、抗性基因序列、多克隆位点序列、启动子序列和Cplx2基因cDNA序列；所述多克隆位点包括EcoR I酶切位点和Spe I酶切位点，所述Cplx2基因cDNA序列包括EcoR I酶切位点、Cplx2基因编码序列和Spe I酶切位点，所述Cplx2基因cDNA序列正向插入所述多克隆位点序列中。
采用上述技术方案，本发明提供的Cplx2基因cDNA序列插入pLVX-IRES-Puro表达载体构建得到的慢病毒表达载体具有转染效率高，用量少，可持续、高效、稳定地提高Cplx2基因表达的优点，可作为有力工具应用于制备治疗Cplx2基因表达对阿尔兹海默症等疾病药物的研究和开发中。
作为本发明的进一步改进，所述Cplx2基因编码序列通过PCR扩增获得，PCR引物包括上游引物和下游引物，所述上游引物的序列为：5’-GCGAATTCATGGACTTCGTCATGAAGCAG-3’，即SEQ ID NO：1，所述下游引物的序列为：5’-GACTAGTTTACTTCTTGAACATGTCCTGC-3’，即SEQ ID NO：2。采用上述PCR引物序列，通过PCR可以扩增出Cplx2基因编码序列，并可成功插入至pLVX-IRES-Puro表达载体中持续表达Cplx2基因，减少了序列合成费用，成本较低。
相应的，本发明还提供特异促进Cplx2基因高表达的慢病毒表达载体的构建方法，包括如下步骤：
A)Cplx2基因引物设计：根据Cplx2基因编码序列，使用Oligo 7分析后选取5’-GCGAATTCATGGACTTCGTCATGAAGCAG-3’，即SEQ ID NO：1作为上游引物，选取5’-GACTAGTTTACTTCTTGAACATGTCCTGC-3’，即SEQ ID NO：2作为下游引物，然后合成所述上游引物和所述下游引物；所述上游引物和所述下游引物无引物二聚体，且退火温度差距较小；
B)Cplx2基因cDNA序列的获得：用所述上游引物和所述下游引物进行PCR扩增，获得大量Cplx2基因编码序列，然后将该序列进行加A尾反应后，用T4DNA连接酶连接到pGM-T载体上得到连接产物，将该连接产物转化到感受态大肠杆菌DH5α中，均匀涂布到含氨苄青霉素LB培养基平板上，挑取阳性单克隆菌落培养保存菌液并进行PCR初步鉴定，将初步鉴定结果说明Cplx2基因cDNA序列插入 成功的菌液进行测序鉴定；用液体LB培养基培养测序鉴定正确的大肠杆菌，并抽提其中带Cplx2基因cDNA序列的pGM-T载体，用限制性内切酶EcoR I酶和Spe I酶双酶切，电泳、切胶回收500bp左右的片段，此片段即为Cplx2基因cDNA序列；
C)
特异促进Cplx2基因高表达的慢病毒载体的构建和鉴定：提取质粒pLVX-IRES-Puro，用限制性内切酶EcoR I酶和Spe I酶双酶切，电泳、切胶回收载体，再用T4DNA ligase将所述Cplx2基因cDNA序列连接到pLVX-IRES-Puro表达载体中，得到连接产物，将该连接产物转化到感受态大肠杆菌DH5α中，均匀涂布到含氨苄青霉素LB培养基平板上，挑取阳性单克隆菌落培养保存菌液并进行PCR初步鉴定，将初步鉴定结果说明Cplx2基因cDNA序列插入成功的菌液进行测序鉴定；
D)
特异促进Cplx2基因高表达的慢病毒载体的抽提：将测序结果证实Cplx2基因cDNA序列插入成功的菌液扩增培养，对重组质粒进行抽提，得到特异促进Cplx2基因高表达的慢病毒表达载体。
本发明利用基因工程技术构建特异促进Cplx2基因高表达的慢病毒表达载体，经鉴定构建成功后，包装成病毒转导入RGC5细胞，嘌呤霉素筛选细胞后，使用实时荧光定量PCR和Western Blot技术分别从mRNA和蛋白水平验证Cplx2基因表达的变化，实验结果证明本发明提供的Cplx2基因cDNA序列成功插入至pLVX-IR ES-Puro表达载体中，能特异、持续、高效、稳定地促进Cplx2基因高表达。
本发明还提供特异促进Cplx2基因高表达的慢病毒表达载体在制备治疗Cplx2基因表达异常相关疾病的药物中的用途。
发明的有益效果
有益效果
本发明提供的特异促进Cplx2基因高表达的慢病毒表达载体具有转染效率高，用量少，能特异、持续、高效、稳定地促进Cplx2基因高表达的优点，可作为有力工具应用于与Cplx2相关的药物研究和开发中；本发明还提供了特异促进Cplx2基因高表达的慢病毒表达载体的构建方法，操作效果好，减少了序列合成费用 ，成本较低。
对附图的简要说明
附图说明
图1为pLVX-IRES-Puro表达载体的质粒图谱。
图2为嘌呤霉素筛选细胞后荧光定量PCR检测结果示意图。
实施该发明的最佳实施例
本发明的最佳实施方式
下面结合附图与具体实施例对本发明做进一步的说明。
RGC5细胞购自上海生命科学院细胞资源中心，293FT细胞购自Thermo Fisher公司，Premix PrimeSTAR HS酶、慢病毒表达载体pLVX-IRES-Puro、病毒包装辅助试剂盒、Lenti-X GoStix试剂盒均购自Takara公司，RNeasy Mini Kit购自QIAGEN公司，pGM-T载体购自天根公司，Endo-Free Plasmid Mini Kit II购自Omega bio-tek公司。
实施例一Cplx2基因引物的设计。
根据Cplx2基因编码序列(GenBank NM_001008220.1)，使用Oligo7对其进行分析，寻找上游引物和下游引物(要求尽可能无引物二聚体且退火温度差距较小)，然后在上游引物和下游引物的5’端分别加入保护碱基与酶切位点EcoR I和EcoR I，设计得到的引物序列如表1所示。设计的PCR引物由上海生工生物工程技术服务有限公司合成。
表1 Cplx2基因的PCR引物序列
[Table 1]
Figure PCTCN2016086064-appb-000001
实施例二特异促进Cplx2基因高表达的慢病毒载体的构建
将合成的引物稀释后，用Premix PrimeSTAR HS酶对Cplx2基因的编码序列进行扩增，电泳回收后然后将其进行加A尾反应后，用T4DNA连接酶连接到pGM-T载体上得到连接产物(Cplx2-T载体)，将该连接产物转化到感受态大肠杆菌DH5α中，均匀涂布到含氨苄青霉素LB培养基平板上，于37℃培养12h，同时设置阴性对照组1(将感受态细胞均匀涂布在不含氨苄青霉素的平板上)、阴性对照组2(将感受态细胞均匀涂布在含100μg/ml氨苄青霉素的平板上)、阳性对照组1(将双酶切空载体的连接产物均匀涂布在含100μg/ml氨苄青霉素的平板上)、阳性对照组2(将空载体均匀涂布在含100μg/mL氨苄青霉素的平板上)。实验组长出了单菌落，阴性对照组1长出了菌落；阴性对照组2、阳性对照组1、阳性对照组2没长出菌落。
从实验组中挑取8个单菌落培养保存后，各取0.5μL培养液，用Cplx2基因的引物进行PCR扩增来初步鉴定，结果表明8个单菌落的培养液均能成功扩增出Cplx2基因，接着将重组载体送至上海生工公司测序。
取测序结果正确的菌，置于液体LB培养基中培养14h，然后提取包含Cplx2基因序列的重组T载体，将其和pLVX-IRES-Puro载体分别先用EcoR I酶和Spe I酶进行双酶切，电泳回收，并用T4DNA连接酶连接回收产物用，再次转化到感受态大肠杆菌DH5α中，均匀涂布到含氨苄青霉素LB培养基平板上，于37℃培养12h，同时设置阴性对照组1(将感受态细胞均匀涂布在不含氨苄青霉素的平板上)、阴性对照组2(将感受态细胞均匀涂布在含100μg/ml氨苄青霉素的平板上)、阳性对照组1(将双酶切空载体的连接产物均匀涂布在含100μg/ml氨苄青霉素的平板上)、阳性对照组2(将空载体均匀涂布在含100μg/mL氨苄青霉素的平板上)。实验组长出了单菌落，阴性对照组1长出了菌落；阴性对照组2、阳性对照组1、阳性对照组2没长出菌落。
从实验组中挑取6个单菌落培养保存后，各取0.5μL培养液，用Cplx2基因的引物进行PCR扩增来初步鉴定。结果表明全部6支培养液均能成功扩增出Cplx2基因，接着将这些重组载体菌液送至上海生工公司测序，测序结果与预期完全相符，获得pLVX-Cplx2质粒。
取出之前保存的重组质粒菌液，取20μL接种到15ml LB培养基(含100μg/ml氨苄青霉素)中，37℃，300rpm培养16h，用Endo-Free Plasmid Mini Kit II进行抽提重组质粒pLVX-Cplx2，测其纯度和浓度，结果如表2所示。
表2 重组质粒的纯度和浓度
[Table 2]
重组质粒 A260/A280 浓度(mg/mL)
pLVX-Cplx2 1.82 627.3
实施例三慢病毒包装
培养293FT细胞，取生长状态良好的细胞接种到六孔中，每孔100000个细胞，用慢病毒包装辅助试剂盒，取抽提的重组质粒pLVX-Cplx2 2μg转染到293FT细胞，48h后收集含病毒的上清培养基，用0.45μm的筛子过滤病毒液，用于感染RGC5细胞，Lenti-X GoStix试剂盒检测病毒的滴度为5000000～50000000IFU。
实施例四慢病毒转导RGC5细胞
接种RGC5细胞于6孔板中，每孔1000000个细胞，12h后细胞密度约为50％，分别取病毒液，用DMEM完全培养基10倍稀释病毒，再加入聚凝胺(polybrene)至终浓度为8μg/mL。去6孔板中的培养基，加入含病毒的DMEM完全培养基(含10％胎牛血清)，24h后弃去含病毒的DMEM完全培养基，更换新鲜的DMEM完全培养基，24h后用0.5μg/ml浓度的嘌呤霉素筛选细胞。筛选10d，每隔3d更换培养基一次，并不断的增加嘌呤霉素的浓度至1.00μg/ml。
实施例五荧光定量PCR检测Cplx2基因表达量。
根据GAPDH和Cplx2基因mRNA序列，利用引物设计软件Oligo 7.0设计引物。
[Table 3]
基因和引物 引物序列(5’-3’)
GAPDH-F TCTGACTTCAACAGCGACACC
GAPDH-R CTGTTGCTGTAGCCAAATTCGT
Cplx2-F GACCCCGACGCGCAGAAAAAGG
Cplx2-R CTGCTTTCTCCTCTGCTTCCTTC
分别接种RGC5细胞、pLVX空载体对照RGC5细胞组、pLVX-Cplx2高表达细胞至6孔板。细胞密度达到80％-90％时，用RNeasy Mini Kit提取各组细胞的总RNA，利用PrimeScrip RT reagent Kit将mRNA逆转录为cDNA，逆转录条件：37℃，15min；85℃，5s；4℃，∞。反转录结束后，加入90μL的RNase Free dH20稀释cDNA，-20℃保存，以便后面检测使用。取各组细胞的cDNA 1μl为模板，以GAPDH为内参，实时荧光定量PCR(QPCR)检测Cplx2相对表达量，设置反应条件：95℃30s，1循环，54℃30s 40循环，95℃5s，60℃1min，95℃15s，利用SYBR Primescript RT-PCR Kit检测各组细胞Cplx2基因相对表达量。将pLVX-Cplx2细胞连续培养20代后，重复以上实验。汇总后的结果如图2所示。可以看到，不管是刚筛选完，还是已经培养20代后的pLVX-Cplx2细胞，其Cplx2基因的表达量较RGC5细胞都有150倍左右的升高，而pLVX空载体细胞的Cplx2基因表达量与RGC5细胞相比基本没有变化，说明本发明提供的Cplx2基因cDNA序列成功插入至pLVX-IRES-Puro表达载体中，能特异、持续、高效、稳定地促进Cplx2基因高表达。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。
工业实用性
本发明提供的特异促进Cplx2基因高表达的慢病毒表达载体具有转染效率高， 用量少，能特异、持续、高效、稳定地促进Cplx2基因高表达的优点，可作为有力工具应用于与Cplx2相关的药物研究和开发中；本发明还提供了特异促进Cplx2基因高表达的慢病毒表达载体的构建方法，操作效果好，减少了序列合成费用，成本较低。

Claims (4)

  1. 一种特异促进Cplx2基因高表达的慢病毒表达载体，其特征在于：包括pLVX-IRES-puro表达载体的基本序列、抗性基因序列、多克隆位点序列、启动子序列和Cplx2基因cDNA序列；所述多克隆位点包括EcoR I酶切位点和Spe I酶切位点，所述Cplx2基因cDNA序列包括EcoR I酶切位点、Cplx2基因编码序列和Spe I酶切位点，所述Cplx2基因cDNA序列正向插入所述多克隆位点序列中。
  2. 根据权利要求1所述的特异促进Cplx2基因高表达的慢病毒表达载体，其特征在于：所述Cplx2基因编码序列通过PCR扩增获得，PCR引物包括上游引物和下游引物，所述上游引物的序列为：5’-GCGAATTCATGGACTTCGTCATGAAGCAG-3’，即SEQ ID NO：1，所述下游引物的序列为：5’-GACTAGTTTACTTCTTGAACATGTCCTGC-3’，即SEQ ID NO：2。
  3. 根据权利要求2所述的特异促进Cplx2基因高表达的慢病毒表达载体的构建方法，其特征在于：包括如下步骤：
    A)Cplx2基因引物设计：根据Cplx2基因编码序列，使用Oligo 7分析后选取5’-GCGAATTCATGGACTTCGTCATGAAGCAG-3’，即SEQ ID NO：1作为上游引物，选取5’-GACTAGTTTACTTCTTGAACATGTCCTGC-3’，即SEQ ID NO：2作为下游引物，然后合成所述上游引物和所述下游引物；
    B)Cplx2基因cDNA序列的获得：用所述上游引物和所述下游引物进行PCR扩增，获得大量Cplx2基因编码序列，然后将该序列进行加A尾反应后，用T4 DNA连接酶连接到pGM-T载体上得到连接产物，将该连接产物转化到感受态大肠杆菌DH5α中，均匀涂布到含氨苄青霉素LB培养基平板上，挑取阳性单克隆菌落培养保存菌液并进行PCR初步鉴定，将初步鉴定结果说明Cplx2基因cDNA序列插入成功的菌液进行测序鉴定；用液体LB培养基培养测序鉴定正 确的大肠杆菌，并抽提其中带Cplx2基因cDNA序列的pGM-T载体，用限制性内切酶EcoR I酶和Spe I酶双酶切，电泳、切胶回收500bp左右的片段，此片段即为Cplx2基因cDNA序列；
    C)特异促进Cplx2基因高表达的慢病毒载体的构建和鉴定：提取质粒pLVX-IRES-Puro，用限制性内切酶EcoR I酶和Spe I酶双酶切，电泳、切胶回收载体，再用T4 DNA ligase将所述Cplx2基因cDNA序列连接到pLVX-IRES-Puro表达载体中，得到连接产物，将该连接产物转化到感受态大肠杆菌DH5α中，均匀涂布到含氨苄青霉素LB培养基平板上，挑取阳性单克隆菌落培养保存菌液并进行PCR初步鉴定，将初步鉴定结果说明Cplx2基因cDNA序列插入成功的菌液进行测序鉴定；
    D)特异促进Cplx2基因高表达的慢病毒载体的抽提：将测序结果证实Cplx2基因cDNA序列插入成功的菌液扩增培养，对重组质粒进行抽提，得到特异促进Cplx2基因高表达的慢病毒表达载体。
  4. 根据权利要求1至3中任一项所述的特异促进Cplx2基因高表达的慢病毒表达载体在制备治疗Cplx2基因表达异常相关疾病的药物中的用途。
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