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Recombinant gland related virus expressing human kallikrein, its preparation method and application

A kallikrein and adenovirus technology, applied in virus/phage, recombinant DNA technology, medical preparations containing active ingredients, etc., can solve the problems of complex replication and transcription regulation of adeno-associated virus

Inactive Publication Date: 2003-04-23
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 3) The replication and transcriptional regulation of adeno-associated virus is quite complicated. According to the co-transfection with or without helper virus, there are two expression modes: proliferative and latent (see attached image 3 shown)

Method used

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  • Recombinant gland related virus expressing human kallikrein, its preparation method and application
  • Recombinant gland related virus expressing human kallikrein, its preparation method and application
  • Recombinant gland related virus expressing human kallikrein, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Cloning of human kallikrein (HK) cDNA and its recombination and construction with rAAV.

[0035] 1) Cloning of human kallikrein (HK) cDNA

[0036] Design primers: P1, 5'-GCAGAATTCATGTGGTTCCTGG-3'; P2, 5'-GACAAGCTTTTACTGGGGGTA-3'. Synthesized by Wuhan Biosynthesis Company. Total RNA was extracted from human liver specimens with a TRIZOL kit (purchased from GIBCO), and subjected to RT-PCR (the kit was purchased from TAKARA, Japan). The PCR instrument was purchased from TECONE, UK. and the product with pXXUF 1 connected. HKcDNA pXXUF 1 replaced by the gfp gene in pXXUF 1 Plasmid composition of -HK, as attached Figure 7 shown.

[0037] The HKcDNA was sequenced, and the results obtained were the same as those from GeneBank. But according to the principle of Kozak sequence: First, the first ATG downstream of the promoter is often used as a translation initiation codon, followed by the 3 positions adjacent to ATG are usually A / G, and the +4 position is G, any 3 or +4...

Embodiment 2

[0053] Antihypertensive Effect of rAAV-HK on Spontaneously Hypertensive Rat (SHR) Model

[0054] The spontaneously hypertensive rat model was purchased from the Shanghai Institute of Hypertension. A total of 12 rats were divided into two groups, and rAAV-HK and rAAV-LacZ viruses were injected through the tail vein respectively, and the amount used for each rat was 1×10 11 p.f.u. Blood pressure was measured weekly after injection, and 24-hour urine was collected every half month. It was found that the blood pressure of the rats in the experimental group began to drop 3-4 weeks after the injection, and all the animals were killed after five months. The results are shown in Figure 9 .

Embodiment 3

[0056] The role of rAAV-HK in preventing stroke

[0057] 8-16 weeks after gene injection, 2 out of 6 animals in the control group (rAAV-lacZ group) had stroke (confirmed by autopsy), one of which was cerebral hemorrhage and the other was multiple cerebral infarction. In the experimental group, none had stroke.

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Abstract

The present invention provides a recombinant glandular related virus capable of expressing human kallikrein, its preparation method and application, and medicine composition containing said recombinant glandular related virus. Said recombinant glandular related virus can drive the human kallikrein gene to make long-period expression in human body, and possesses good physiological and pathologic physiological action for effectively reducing blood pressure, improving renal function and preventing apoplexy, and can be used for clinical curing the above-mentioned diseases.

Description

technical field [0001] The invention relates to the fields of gene recombination and gene therapy, in particular to a recombinant adeno-associated virus vector and human kallikrein gene recombination, a preparation method and application of the recombination, and a pharmaceutical composition containing the recombination. Background technique [0002] Hypertension is a chronic disease influenced by multiple factors and originating from multiple genes. Currently, the only effective way to treat this disease is to take antihypertensive drugs for a long time, or even for life. Burden, long-term use of antihypertensive drugs will also have some adverse effects on the machine. Therefore, exploring gene therapy methods and drugs for hypertension has become an important topic at present. [0003] Human kallikrein (human kallikrein, HK) is a kind of serine protease, which can cleave kininogen to generate vasoactive peptide-kinin. Kinins can promote the transp...

Claims

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Application Information

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IPC IPC(8): A61K39/235A61P9/12C12N7/01C12N15/34C12N15/79
Inventor 汪道文李宏伟王涛
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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