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Design and application of PCSK9 targeting recombinant vaccine

An immune response and composition technology, applied in the field of PCSK9-targeted recombinant vaccine design, can solve problems such as muscle toxicity, elevation of liver transaminases, and adverse reactions

Active Publication Date: 2015-11-25
SHANGHAI HYCHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Among these many lipid-lowering drugs, statins have the best therapeutic effect and are the most widely used at present, but the use of these drugs will also produce certain adverse reactions. For example, large doses of statins can cause muscle toxicity and increase liver transaminases. Fibrates and niacin may cause gastrointestinal discomfort, liver and kidney damage, etc.
Moreover, some patients are intolerant to statins, more importantly, most of the high-risk and extremely high-risk patients with cholesterol, such as severe primary dyslipidemia (such as familial hypercholesterolemia; familial combined hyperlipidemia or beta lipoprotein Abnormal) patients; patients with very high basal LDL-C levels; patients at very high CVD risk who require intensive treatment; statin therapy due to potentially serious drug interactions, comorbidities, or intolerance to high-dose statins Dose-limited patients, etc., whose LDL-C target value cannot be reached by existing drug therapy

Method used

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  • Design and application of PCSK9 targeting recombinant vaccine
  • Design and application of PCSK9 targeting recombinant vaccine
  • Design and application of PCSK9 targeting recombinant vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0167] Embodiment 1 Recombinant plasmid construction and fusion protein expression

[0168] 1.1 Design the fusion protein (recombinant vaccine) according to Table 2.

[0169] Table 2 Design of fusion protein

[0170]

[0171] 1.2 Plasmid construction and fusion protein expression

[0172] 1.2.1 Experimental reagents

[0173] ExTaq enzyme, 10×ExTaqbuffer, dNTPs (Takara), 10×DNA loading buffer (Takara), KOD enzyme (TOYOBO), 10×KODplusbuffer, MgSO 4 (25mmol / L), dNTPs (TOYOBO Company), Gold-view dyeing solution (SBS Race Parkson Company), DNAMarker (TIANGEN Biological Co., Ltd.).

[0174] 1.2.2 Experimental method

[0175] 1.2.2.1 Primer design and synthesis

[0176] (1) Nested primer design

[0177] Nested primers were designed according to the accession numbers of human and mouse PCSK9 gene mRNA sequences provided by GenBank (GenBank: EF692496.1, NM_153565.2), and the primers were named hF1, hR1, hF2, hR2, mF1, mR1, mF2, mR2. See Appendix 1.

[0178] (2) Fusion primer...

Embodiment 2

[0222] Example 2 Purification of Fusion Protein and Refolding of Inclusion Body

[0223] 2.1 Experimental steps

[0224] Use the conventional method to ferment the genetically engineered strain obtained in Example 1. After the fermentation is completed, it is ultrasonically crushed, and the target protein is affinity purified using a GST column. The inclusion body of PCSK9-8 is refolded and identified by SDS-PAGE electrophoresis. , BCA protein quantification method was used for protein quantification.

[0225] Concentrate the purified target protein with an ultrafiltration tube with a pore size of 3kD or 10kD, and adjust the protein concentration to 0.5-1mg / mL with 1×PBS. In the ultra-clean bench, the prepared protein solution was divided into 1.5mLEP tubes, and 15% glycerol was added, and stored in a -70°C refrigerator for later use.

[0226] 2.2 Experimental results

[0227] 2.2.1 Affinity purification of fusion protein

[0228] The size of PCSK9-1, PCSK9-2, PCSK9-3 and ...

Embodiment 3

[0239] Embodiment 3 animal immunization and titer determination

[0240] 3.1 Experimental materials and instruments

[0241] 3.1.1 Experimental reagents

[0242] mPCSK9 recombinant protein, hPCSK9 recombinant protein (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.); secondary antibody (HRP-labeled goat anti-mouse antibody), pre-stained marker, easyWesternProteinLader (purchased from GenScript Nanjing Biotechnology Co., Ltd.)

[0243] 3.2 Experimental method

[0244] 3.2.1 Mice immunization method

[0245] The animals used for immunization were female BALB / c mice aged 6-7 weeks at SPF level (purchased from Beijing Weitong Lihua), which were raised in the ultra-clean room of the Animal Experiment Center of Shanghai Jiaotong University. They were randomly divided into 8 groups, with 6 mice in each group. 10mMPBS, DTT, PCSK9-1, PCSK9-2, PCSK9-3, PCSK9-6, PCSK9-7 and PCSK9-8 were used as antigens respectively. Immunization was carried out on days 0, 7, and 14, ...

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Abstract

The present invention discloses design and application of a PCSK9 targeting recombinant vaccine, and specifically discloses a PCSK9 targeting recombinant vaccine. Experimental results show that the recombinant vaccine can effectively induce animal body to produce immunoreaction aiming at PCSK9, and effectively reduce cholesterol level. The invention also provides an antigen epitope peptide and fusion protein based on the PCSK9.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to the design and application of PCSK9 targeting recombinant vaccine. Background technique [0002] Atherosclerosis (AS) is a harmful state caused by the deposition of fat, thrombus, connective tissue and calcium carbonate in blood vessels (mainly arteries), which can lead to myocardial ischemia, myocardial infarction, Cardiovascular and cerebrovascular diseases such as stroke and peripheral vascular disease have seriously affected human health and quality of life. Cardiovascular disease (CVD) caused by atherosclerosis is currently the leading cause of death in developed countries ahead of cancer. Although CVD mortality has shown a downward trend with the development of medical technology in recent years, data show that CVD is still the leading cause of death in developed regions. According to statistics, in 2005, there were 278.9 deaths from CVD per 100,000 Ameri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/64C12N15/62C12N15/57A61K39/00A61K47/48A61K48/00A61P3/06A61P9/10
Inventor 李荣秀陈凌祝先潮
Owner SHANGHAI HYCHARM
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