Bacterin of nucleic acid for anti atherosclerosis and preparation method

A technology for atherosclerotic nucleic acid and nucleic acid vaccines, which is applied in pharmaceutical formulations, antibody medical ingredients, and medical preparations containing active ingredients, etc., and can solve the problem of patients' lack of long-term compliance, long-term use, diet and exercise are not always effective And other issues

Inactive Publication Date: 2005-01-12
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among the currently used cholesterol-lowering methods, dietary restriction is the most common, but diet and exercise are not always effective due to lack of long-term compliance or genetic predisposition to high LDL level...

Method used

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  • Bacterin of nucleic acid for anti atherosclerosis and preparation method
  • Bacterin of nucleic acid for anti atherosclerosis and preparation method
  • Bacterin of nucleic acid for anti atherosclerosis and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: Eukaryotic expression vector pCR3.1-X 8 - Construction of HBc

[0056] Genomic RNA of HBV was extracted from serum of HBcAg positive patients in Nanjing Second Hospital with QIAamp DNA Blood Mini Kit. The random primers sold in the market are used to reverse transcribe the RNA into DNA, and two pairs of primers P1-P4 that can be complementary to the gene are synthesized respectively according to the sequence of the hepatitis B core antigen gene. The 5' ends of primers P1, P3, and P4 contained HindIII, AgeI, and XbaI restriction sites, respectively, while the 5' end of primer P2 contained two restriction sites, PstI and AgeI, respectively. In the first step, use P1 and P2 as primers, and use the HBV DNA obtained by reverse transcription as a template to carry out PCR, 94°C, 5min; 94°C, 1min, 58°C, 1min, 72°C, 1min, a total of 30 rounds; 72°C , 10min. The N-terminus of the amplified PCR product, namely HBc, was digested with restriction endonucleases Hind...

Embodiment 2

[0067] Example 2 Anti-atherosclerosis nucleic acid vaccine recombinant plasmid pCR3.1-X 8 - Construction of HBc-CETPC

[0068] According to the amino acid sequence of the C-terminal peptide of the cholesteryl ester transfer protein, the complete sequence of the C-terminal polypeptide gene of the cholesteryl ester transfer protein is designed with the aid of a computer. Based on the sequence, three primers were designed and chemically synthesized. Firstly, the partial sequence of the cholesteryl ester transfer protein gene was synthesized by PCR method, and then the full sequence of the C-terminal peptide gene of the cholesteryl ester transfer protein was obtained by the second PCR amplification. Specifically, the first step: primers C1 and C2 are mixed in a certain ratio, and the two are primers and templates for each other, 95°C, 1min, 55°C, 1.5min, 72°C, 2min, a total of 10 rounds; 72°C, 10min. Using the PCR product as a template, PCR amplification was carried out with C1 ...

Embodiment 3

[0074] Example 3 Engineering Bacteria Fermentation and Large-Scale Preparation of Anti-Atherosclerosis Nucleic Acid Vaccine Recombinant Plasmid

[0075] pCR3.1-X 8 -The engineering bacteria culture of the HBc-CETPC plasmid was carried out in a 1000ml shake flask. Pick a single colony from a freshly transferred plate and insert it into LB liquid medium, cultivate it at 37°C for 8 hours to the logarithmic phase as a first-level seed, and insert it into a 1000ml shaker containing 250ml LB liquid medium at an inoculum size of 1 / 250. In the flask, culture was continued for 12 hours at 37°C with vigorous shaking. Collect the thalli by centrifugation at 5000rpm for 15min, fully resuspend the bacterial sediment in 9ml solution I [50mmol / L glucose, 25mmol / L Tris-HCl (pH8.0), 10mmol / L EDTA (pH8.0)], then add 100μl RNase solution [10mg / ml RNase, 10mmol / L Tris-HCl (pH7.5), 15mmol / L NaCl], mix well and place in ice bath for 10min. Slowly add 20ml of freshly prepared solution II [0.2mmol...

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Abstract

A nucleic acid vaccine for preventing and treating atherosolerosis and its preparing process are disclosed. The core protein of hepatitis B is used as its immune carrier to intensify the immunogenicity of CETPC. The body immunized by said vaccine can generate CETPC antibody to suppress the activity of cholesteryl ester transfer protein, so decreasing the generation of atherosclerosis spots. It can also act on the formed atherosclerosis sports to relay them.

Description

technical field [0001] In the medical field, the anti-atherosclerosis nucleic acid vaccine provided by the invention can be used for preventing and treating atherosclerosis. Background technique [0002] Atherosclerosis (AS) is a common vascular disease caused by degenerative and proliferative lesions of the arteries, resulting in thickening of the vessel wall, loss of elasticity and narrowing of the lumen. Thin and complex carbohydrates are deposited, accompanied by smooth muscle cells and fibrous tissue proliferation, and then the intima and media gradually degenerate and calcify. The disease mainly involves large muscular elastic arteries (such as the aorta) and medium-sized muscular elastic arteries (coronary arteries and cerebral arteries are the most affected). Functional disorders, among which coronary heart disease caused by coronary atherosclerosis, that is, coronary heart disease, is the most common type of organ lesions caused by the disease. The incidence rate ...

Claims

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Application Information

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IPC IPC(8): A61K38/16A61K39/29A61P9/10
Inventor 刘景晶茅丹朱政吴洁曹荣月宗莉
Owner CHINA PHARM UNIV
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