Preparation method and application of capsid-protein-mutant double-stranded recombinant adeno-associated virus containing mediated-membrane-stabile CD40L gene

A CD40L-M, 5-Y719F-CD40L-M technology, applied in the field of double-stranded recombinant adeno-associated virus vector and its preparation, can solve the problem of less than 15% survival rate, and achieves inhibition of lung cancer growth, promotion of cell apoptosis, The effect of inducing antitumor immunity

Inactive Publication Date: 2013-04-24
JIANGSU PROVINCE HOSPITAL
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  • Abstract
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Problems solved by technology

Although some progress has been made in the early diagnosis and comprehensive treatment of lung cancer in recent years, 90% of lung cancers have metastasized when they are discovered, and the 5-year survival rate is less than 15%.
[0011] At present, there is no report at home and abroad which serotype of scAAV with a mutation in the tyrosine residue of the coat can efficiently transduce lung cancer cells, and there is no report on the construction of an adeno-associated virus vector with a membrane-stable CD40L gene and its application in anti-tumor gene therapy report

Method used

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  • Preparation method and application of capsid-protein-mutant double-stranded recombinant adeno-associated virus containing mediated-membrane-stabile CD40L gene
  • Preparation method and application of capsid-protein-mutant double-stranded recombinant adeno-associated virus containing mediated-membrane-stabile CD40L gene
  • Preparation method and application of capsid-protein-mutant double-stranded recombinant adeno-associated virus containing mediated-membrane-stabile CD40L gene

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Experimental program
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Embodiment 1

[0059] Such as Figure 1A with Figure 1B , pcDNA3.1 + - CD40L-M construction and identification. The specific steps are: ①pcDNA3.1 + - CD40L plasmid construction and identification. Isolation of human mononuclear cells from peripheral blood; extraction of total cellular RNA and cDNA transcription; RT-PCR amplification of the target gene CD40L (upstream primer: 5'-CCGGAATTCCCAGAAGATA-3', downstream primer: 5'CCGCTCGAGTCAGCTCCA-3', product size: 850bp); the target gene fragment and pcDNA3.1 were digested with restriction enzymes EcoRI and XhoI + Vector (purchased from Invitrogen) ligation; pcDNA3.1 +-CD40L质粒转化和鉴定。②pcDNA3.1 + -CD40L-M质粒构建和鉴定。以源克隆pcDNA3.1 + -CD40L设计并合成引物引入变异点(Q 114 K 115 D. 117 Q 118 N 119 for P 114 R 115 E. 117 E. 118 D. 119 );高保真DNA聚合酶platinum pfx DNA Polymerase(Invitrogen)进行PCR扩增;对PCR产物进行末端平滑及5’端磷酸化反应,然后进行自身连接反应,产物转化感受态细胞DH5a;挑选克隆鉴定。将pcDNA3.1 + -CD40L和pcDNA3.1 + -CD40L-M两种重组质粒经限制性内切酶EcoR I和Xho I双酶切鉴定,1%琼脂糖凝胶电泳显示为约850bp和4800bp的两个片段( Figure 1A...

Embodiment 2

[0061] Such as Figure 2A with 2B ,pdsAAV-CB-CD40L-M质粒构建及鉴定。具体步骤为:①扩增pcDNA3.1 + -CD40L-M及pdsAAV-CB-GFP,引物为T7和T405-R(T405-R:CTTAGGAGCTCGTCGACGTCGAGGCTGATCAGCGGGT),CD405-R引物添加了Sac I酶切位点及保护碱基;然后用BamHI和Sac I双酶切PCR产物,克隆到目的载体中pdsAAV-CB-GFP中;挑单克隆菌检,阳性克隆测序,得到与标准序列一致的克隆。

Embodiment 3

[0063] Such as Figure 3A ,不同血清型AAV外壳表面约730位点的碱基序列及AAV2 / 5突变位点构建。 对AAV2和AAV2 / 5基因行同源比对及晶体结构分析,确定AAV5衣壳蛋白表面719位点的氨基酸分别对应于AAV2衣壳蛋白730位点的酪氨酸残基。 use II Site-Directed Mutagenesis Kit(Alignment Technologies)标准点突变试剂盒。先合成突变链:DNA模板变性,与包含所需突变的突变引物退火,使用 DNA聚合酶进行引物延生,构建 AAV2 / 5病毒外壳蛋白719位点酪氨酸(Y)编码碱基点突变为苯丙氨酸(F)编码碱基的pAAV2 / 5-Y719F-R / C质粒(引物AAV2 / 5-Y719-F:CCCATTGGCACCAGATTCCTGACGCGTAATCTGTAATTGCTTG,AAV2 / 5-Y719-R:CAAGCAATTACAGATTACGCGTCAGGAATCTGGTGCCAATGGG)。Dpn I消化甲基化和半甲基化亲本DNA模板,即pAAV2 / 5-R / C;将突变的分子转化进感受态细胞修复切口;筛选阳性克隆,送测序并测全长,得到pAAV2 / 5-Y719F-R / C质粒。

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Abstract

The invention discloses a nucleotide sequence and an amino acid sequence of a human derived membrane-stabilization mutant gene CD40L-M, and a plasmid carrier pdsAAV-CB-CD40L-M. The invention further discloses a recombinant adeno-associated virus scAAV2/5-Y719F-CD40L-M using CD40L-M as a target gene and highly-efficiently transducing lung cancer cells, a preparation method thereof, and an application thereof in preparing anti-cancer drugs. In vitro and in vivo tests of transduction of the CD40L-M gene into the lung cancer cells show that generation of the sCD40L is substantially reduced, a traditional AAV transduction efficiency is improved by using the recombinant adeno-associated virus scAAV2/5-Y719F-CD40L-M, the lung cancer cells can be highly-efficiently transduced, and the CD40L-M gene induces retardance of cell cycles, promotes cell apoptosis, induces immunization activation, inhibits growth of lung cancer in the body and reduces side effects such as liver and kidney damage.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and in particular relates to a double-chain recombinant adeno-associated virus vector mediating membrane-stabilized CD40L-M gene capsid protein mutation and its preparation method and application. Background technique [0002] Lung cancer is one of the malignant tumors with the highest mortality rate in China and even in the world. Although some progress has been made in the early diagnosis and comprehensive treatment of lung cancer in recent years, 90% of lung cancers have metastasized when they are discovered, and the 5-year survival rate is less than 15%. Therefore, there is an urgent need to find new ways to treat primary lung cancer and its metastases. [0003] At present, immune gene therapy is regarded as one of the most promising tumor treatment methods. Tumor immunity involves systemic immunity and local immunity, including complex processes. The tumor microenvironment (micr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/864C12N15/66C12N7/01A61K48/00A61P35/00C12R1/93
Inventor 吴剑卿赵卫红许伟
Owner JIANGSU PROVINCE HOSPITAL
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