CN109678946B

CN109678946B - Human vascular smooth muscle cell L-type calcium channel immunogenic peptide segment and vaccine and application thereof

Info

Publication number: CN109678946B
Application number: CN201910118277.7A
Authority: CN
Inventors: 廖玉华; 陈霄; 王一逸; 邱志华; 廖梦阳
Original assignee: Wuhan Huajiyuan Biotechnology Development Co ltd
Current assignee: Wuhan Huajiyuan Biotechnology Development Co ltd
Priority date: 2019-02-14
Filing date: 2019-02-14
Publication date: 2022-05-13
Anticipated expiration: 2039-02-14
Also published as: CN109678946A

Abstract

The invention discloses a human vascular smooth muscle cell L-type calcium channel immunogenic peptide fragment, a vaccine and an application thereof, wherein the human vascular smooth muscle cell L-type calcium channel immunogenic peptide fragment is any one of the following four peptide fragments, and the amino acid sequences of the peptide fragments are VPAEDDPSPC, DSSKQTEAECK, DSHTEDKGPI and CAPESEPSNSTE respectively. Coupling and combining the vascular smooth muscle cell L-type calcium channel immunogenic peptide segment with recombinant Qbeta-2 aa phage virus-like particle protein, and repeatedly and orderly arranging on the surface of the virus-like particle to form the polypeptide-vector vaccine. Discloses the application of the vaccine in the aspect of treating essential hypertension. The vaccine can induce and generate high-titer specific antibodies in experimental animals, and can remarkably reduce the blood pressure of spontaneous hypertensive rats.

Description

Human vascular smooth muscle cell L-type calcium channel immunogenic peptide segment and vaccine and application thereof

Technical Field

The invention relates to the field of biotechnology and medicine, in particular to an L-type calcium channel immunogenic peptide fragment of human vascular smooth muscle cells, and a vaccine and application thereof.

Background

Hypertension is a worldwide problem seriously harming human health, and according to the statistical result of 'Chinese cardiovascular disease report 2017', about 2.7 hundred million hypertension, 1300 million stroke, 1100 million coronary heart disease, 450 million heart failure and cardiovascular death rate are the first diseases in our country at present. Despite the use of many chemical drugs for hypertension treatment, poor compliance, low treatment rate (40%) and low control rate (10%) of hypertension treatment in China remain unsolved problems, and thus hypertension complications are high and seriously harm human health. The therapeutic antihypertensive vaccine has the advantages of accurate biological target therapy (high target specificity) and lasting effect (the effect can be maintained for several months after 1-3 months of immunization), and shows good clinical application prospect.

The development of antihypertensive vaccines currently focuses internationally on the Renin Angiotensin System (RAS). The clinical IIa phase experiment of the CYT006-AngQb antihypertensive vaccine (target spot: angiotensin II) developed by the company Cytos of Switzerland in 2007 proves that the vaccine has good safety and can reduce the blood pressure of a hypertensive by about 8 mmHg. According to the analysis of the reason that the CYT006-AngQb antihypertensive vaccine has limited effect, the vaccine generates an antibody aiming at angiotensin II (AngII) after immunization, the antibody plays a role in reducing blood pressure by neutralizing AngII, but the AngII is inevitably neutralized to cause renin activation due to the RAS feedback activation phenomenon, and finally more AngII is generated, so that the antihypertensive effect of the vaccine is weakened. In addition, hypertension is a disease with multiple factors involved, and the control effect of only one therapeutic target on blood pressure is limited. Two patents with application numbers 200610019290.X and ZL201110171715.X in 2006 and 2011 disclose targeted antihypertensive vaccines (ATR12181 and ATRQ beta-001 antihypertensive vaccines) for mouse and human angiotensin II-1 receptor (AT1R), which effectively reduce the blood pressure of Spontaneous Hypertensive Rats (SHRs) and hypertensive mice and remarkably reverse the reconstitution of target organs, and have good safety, and the vaccine has the remarkable characteristic of blocking AT1R and simultaneously activating RAS without feedback.

The selection of proper target and carrier is the key for the successful development of therapeutic vaccine, and the new antihypertensive target is focused on the vascular smooth muscle L-type calcium channel. During the development process of hypertension, the activation of L-type calcium channels of vascular smooth muscle cells plays a vital role, the calcium channels are opened after being activated, extracellular calcium ions enter cells to increase the concentration of intracellular calcium ions, and on one hand, the smooth muscle cells are contracted, especially in arterioles and arterioles to increase vascular resistance and blood pressure. Meanwhile, the increase of the concentration of calcium ions in cells can also promote the expression of various transcription factors and participate in the pathological changes such as vascular remodeling, vascular inflammation and the like. The calcium channel blocker is an effective medicine for treating hypertension, particularly the long-acting calcium channel blocker has definite curative effect and is widely used in clinic, so the L-type calcium channel is an ideal new target of the antihypertensive vaccine.

L-type calcium channels (cav1.2) are composed of α 1c, α 2 δ and β subunits, where the α 1c subunit has 4 domains, each with 6 α transmembrane helices, and where the junction between the 5 th and 6 th helices (i.e., region E3) constitutes the internal mouth of the channel through which calcium ions flow into the cell.

Currently, active or passive immunization methods are not used internationally to block the channel.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an L-type calcium channel immunogenic peptide fragment of human vascular smooth muscle cells for effectively treating essential hypertension, a vaccine and an application thereof;

antibodies directed to the E3 region may have a functional inhibitory effect on potassium channels, sodium channels, P/Q and N-type calcium channels. Through the research on the structure of the L-type calcium channel of the vascular smooth muscle cell, the invention screens the immunogenic peptide fragment and relates to the E3 region sequence of the I, III and IV structural domains of the L-type calcium channel, so as to obtain the immunogenic peptide fragment of the L-type calcium channel of the human vascular smooth muscle cell. The peptide segment and the vaccine vector Qbeta-2 aa phage virus-like particles are prepared into a peptide segment-vector vaccine through chemical coupling, a specific antibody can be induced in vivo after animals are immunized, the blood pressure of a hypertensive rat is remarkably reduced, and the peptide segment-vector vaccine can be applied to treatment of primary hypertension.

The first purpose of the invention is to provide an L-type calcium channel immunogenic peptide fragment of human vascular smooth muscle cells.

In order to realize the first purpose, the invention designs a human vascular smooth muscle cell L-type calcium channel immunogenic peptide fragment, wherein the human vascular smooth muscle cell L-type calcium channel immunogenic peptide fragment is any one of the following four peptide fragments, the amino acid sequences of the human vascular smooth muscle cell L-type calcium channel immunogenic peptide fragment are VPAEDDPSPC, DSSKQTEAECK, DSHTEDKGPI and CAPESEPSNSTE respectively, and the human vascular smooth muscle cell L-type calcium channel immunogenic peptide fragment is shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.

The above VPAEDDPSPC, DSSKQTEAECK, DSHTEDKGPI, CAPESEPSNSTE, distributed in alpha_1CThe E3 region in the I, III, IV domains of the subunits, which is 10-12 amino acids in length, contains as many negatively charged glutamic acid (E) or aspartic acid (D) residues as possible, especially glutamic acid (E) residues, and has been predicted to have good hydrophilicity and immunogenicity by bioinformatics software, and the peptides each contain a first attachment site, either contained in the peptide itself or added by chemical coupling techniques, such as sulfhydryl groups, selected from cysteine residues (C).

Preferably, the amino acid sequence of the L-type calcium channel immunogenic peptide fragment of the human vascular smooth muscle cell is CAPESEPSNSTE.

The second purpose of the invention is to provide a carrier vaccine which comprises one or more than one immunogenic peptide fragment connected with an immunogenic carrier

In order to achieve the second object, the invention designs a human vascular smooth muscle cell L-type calcium channel immunogenic carrier vaccine, which is prepared by coupling the human vascular smooth muscle cell L-type calcium channel immunogenic peptide segment selected from the group consisting of the human vascular smooth muscle cell L-type calcium channel immunogenic peptide segment of claim 1 and a carrier.

Preferably, the human vascular smooth muscle cell L-type calcium channel immunogenic carrier vaccine is prepared by coupling the human vascular smooth muscle cell L-type calcium channel immunogenic peptide fragment CAPESEPSNSTE selected from the group consisting of the above with a carrier.

Preferably, the carrier is Q beta-2 aa phage virus-like particle protein or keyhole limpet hemocyanin.

Combining the peptide fragment with a carrier by using a heterobifunctional cross-linking agent sulfo-SMCC, and repeatedly and orderly arranging the peptide fragment and the carrier on the surface of the virus-like particle to form the peptide fragment-carrier vaccine. The immunogenic peptide segments are displayed and arranged on the surface of the carrier in a certain density to form high-immunogenicity array display, which is beneficial to inducing the generation of high-potency specific anti-human and rat vascular smooth muscle L-type calcium channel immunogenic peptide segment antibodies, remarkably reduces the blood pressure of spontaneous hypertension rats and can be used for treating primary hypertension.

The third purpose of the invention is to provide the application of the vector vaccine in preparing the medicine for treating the primary hypertension.

In order to achieve the third purpose, the invention designs an application of the human vascular smooth muscle cell L-type calcium channel immunogenic carrier vaccine in preparation of a medicament for treating essential hypertension.

The invention has the beneficial effects that:

1. the invention synthesizes human vascular smooth muscle cell L-type calcium channel immunogenic peptide fragments through the research of vascular smooth muscle cell L-type calcium channel structure, which comprise four kinds, wherein the amino acid sequences are VPAEDDPSPC, DSSKQTEAECK, DSHTEDKGPI and CAPESEPSNSTE respectively, and the amino acid sequences are E3 region sequences of I, III and IV structural domains related to the L-type calcium channel; the synthesized peptide segment can cause specific immune response in spontaneous hypertensive rats, and generate specific antibodies aiming at the peptide segment, wherein the antibodies can be specifically combined with L-type calcium channels to block calcium ion inflow, vasoconstriction and blood pressure rise caused by channel opening.

2. The optimized L-type calcium channel immunogenic peptide fragment of vascular smooth muscle cells of the inventor is CAPESEPSNSTE, and the first verification internationally proves that the blood pressure lowering effect of the CAPESEPSNSTE epitope is better, the peptide fragment-vector vaccine prepared by the invention can obviously lower the blood pressure of spontaneous hypertensive rats, and the vaccine prepared by CAPESEPSNSTE has the best effect.

Drawings

FIG. 1 is a gel electrophoresis test image of the vector vaccines Q β -VC10, Q β -DK11, Q β -DI10 and Q β -CE12 in example 2;

in the figure, lane 1 is Qbeta-2 aa phage virus-like particle protein monomer, lane 2 is vaccine Qbeta-VC 10, lane 3 is vaccine Qbeta-DK 11, lane 4 is vaccine Qbeta-DI 10, lane 5 is vaccine Qbeta-CE 12, and lane 6 is protein Marker. And (3) displaying an electrophoresis result: monomers of the Q beta-2 aa virus-like particle can be coupled with 1-4 immunogenic peptide fragments.

FIG. 2 is a graph showing the change in antibody titer; namely Q beta-VC 10, Q beta-DK 11, Q beta-DI 10 and Q beta-CE 12 vaccines are used for immunizing SHRs, the titer of the anti-peptide antibody is changed (the sample is diluted according to the ratio of 1: 5000), and after rats are immunized by each carrier vaccine, the antibody for resisting the L-type calcium channel immunogenic peptide fragment is generated

FIG. 3 is a chart of the trend of the systolic blood pressure changes of SHRs, wherein Control is a PBS Control group, VLP is a Q beta immune group, Q beta-CE 12 is a Q beta-CE 12 vaccine immune group, and Amlodipine is an Amlodipine gastric perfusion group.

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

The following human vascular smooth muscle cell L-type calcium channel immunogenic peptide fragment is abbreviated as follows:

VPAEDDPSPC peptide fragment is hereinafter abbreviated as VC10, as shown in SEQ ID No. 1;

DSSKQTEAECK peptide fragment is hereinafter abbreviated as DK11, as shown in SEQ ID No. 2;

DSHTEDKGPI peptide fragment is hereinafter referred to as DI10, shown as SEQ ID No. 3;

CAPESEPSNSTE peptide fragment is hereinafter abbreviated as CE12, as shown in SEQ ID No. 4.

EXAMPLE 1 preparation of immunogenic peptide fragments of human vascular smooth muscle cell L-type calcium channel

B cell epitope prediction is carried out according to bioinformatics technologies such as amino acid hydrophilicity, antigenicity, sequence space conformation and the like, and 4 immunogenic peptide fragments aiming at the L-type calcium channel are designed: VPAEDDPSPC, DSSKQTEAECK, DSHTEDKGPI, CAPESEPSNSTE are provided. The peptide fragment is synthesized by PSSM-8 type automatic peptide synthesizer (Shimadzu, Japan), and the purity of the synthesized peptide fragment is over 95 percent through high performance liquid chromatography analysis. Freeze-drying the obtained peptide fragment, subpackaging and placing in a freezing tube, and freezing at-80 ℃ for later use.

EXAMPLE 2 preparation of vascular smooth muscle cell L-type calcium channel immunogenic peptide fragment-vector vaccine

The Q beta-2 aa phage virus-like particle protein is used for preparing 4 peptide fragment-vector vaccines Q beta-VC 10, Q beta-DK 11, Q beta-DI 10 and Q beta-CE 12, and the specific preparation process is as follows:

1. preparation of Q β -2aa phage virus-like particle protein: english abbreviations for Q β -2aa bacteriophage virus-like particle proteins are: q β -2aa VLP, hereinafter Q β -2aa bacteriophage virus-like particle protein, is referred to as Q β -2aa VLP. It is prepared by the following method:

1) obtaining a recombinant strain expressing Q β -2aa VLP: the recombinant strain is Escherichia coli DH5 alpha/pGEXQ beta-A1, and the recombinant strain can induce and produce Q beta-2 aa virus-like particle protein. The preservation number of the Escherichia coli DH5 alpha/pGEXQ beta-A1 is CCTCC NO: the specific preparation process of M209282 is shown in Chinese patent: a preparation method and application of Q beta-2 aa phage virus-like particle protein are disclosed as CN 101921733B grant bulletin date 2013.06.05.

2) Induction of expression of Q β -2aa VLP: firstly, taking out a preserved escherichia coli DH5 alpha/pGEXQ beta-A1 recombinant strain from a liquid nitrogen tank, activating the recombinant strain, coating the recombinant strain on an LB solid medium flat plate, culturing overnight in an incubator at 37 ℃, selecting a single colony to culture in an LB liquid medium, culturing for 5 hours in a constant temperature shaking table at 37 ℃, adding 0.2M IPTG (isopropyl-beta-thiogalactoside) to induce the recombinant strain to express Q beta-2 aa VLP, inducing for 6 hours, collecting a bacterial liquid, and performing ultrasonic lysis to obtain a lysis supernatant;

3) purification of Q β -2aa VLP: performing ammonium sulfate precipitation, acidification treatment, hydrophobic chromatography and gel chromatography on the cracked supernatant to obtain purified Q beta-2 aa VLP;

4) identification of Q β -2aa VLP: carrying out decomplexation treatment on the purified Q beta-2 aa VLP by using Dithiothreitol (DTT), identifying the molecular weight of the decomplexed Q beta-2 aa VLP by gel electrophoresis, and observing the form size and the particle size of the decomplexed Q beta-2 aa VLP by using an electron microscope; and finally determining the obtained protein as the Q beta-2 aa VLP through test results.

2. Q beta-VC 10, Q beta-DK 11, Q beta-DI 10 and Q beta-CE 12 vaccines are prepared by coupling peptide segment VPAEDDPSPC, DSSKQTEAECK, DSHTEDKGPI, CAPESEPSNSTE with a vector Q beta virus-like particle by using a heterobifunctional cross-linking agent Sulfo-SMCC (Pierce, MSA), and the steps are as follows:

1) and (3) dissolving 1mg of sulfo-SMCC with the same mass as the Q beta virus-like particles in 10 mu l of DMSO, adding the SMCC solution into Q beta, uniformly blowing, and reacting in a 37 ℃ thermostat for 30 min.

2) Adding the SMCC-Qbeta reaction system into a 100kD centrifuge tube, adding PBS (phosphate buffer solution) with the pH value of 6.8, adding PBS at 20 ℃, 5000Xg for 10-20min, centrifuging for 2-3 times, and filtering to remove the monomer SMCC which is not combined with VLP.

3) Peptide fragment dd H₂O is dissolved to 10mg/ml, and the mass of the peptide segment and the carrier is 1: 2. The peptide fragment is added into a Q beta-SMCC system, and the reaction condition is observed. The mixture was placed on a shaker and reacted at room temperature 1050 times/min for 2 hours.

4) The vaccine was added to a 0.22 μm dialysis membrane, placed in PBS and dialyzed overnight to remove free peptide fragments.

SDS-PAGE electrophoresis to identify Q beta-VC 10, Q beta-DK 11, Q beta-DI 10 and Q beta-CE 12 vaccines. In FIG. 1: the SDS-PAGE electrophoresis result after coupling of the virus-like particle and the peptide fragment shows that the lane 1 is Q beta-2 aa phage virus-like particle protein monomer, the lane 2 is vaccine Q beta-VC 10, the lane 3 is vaccine Q beta-DK 11, the lane 4 is vaccine Q beta-DI 10, the lane 5 is vaccine Q beta-CE 12, and the lane 6 is protein Marker. And (3) displaying an electrophoresis result: monomers of the Q beta-2 aa virus-like particle can be coupled with 1-4 immunogenic peptide fragments.

Example 3 human vascular smooth muscle cell L-type calcium channel immunogenic peptide fragment-vector vaccine antihypertensive effect study

Male Spontaneous Hypertensive Rats (SHR) were purchased from Beijing Wittingle laboratory animal technology, Inc., and were housed in the SPF-level animal laboratory center of the college of medicine, using 12h/12h light and non-restrictive general diet. The method is specifically divided into 7 groups: PBS control group, VLP group, Q beta-VC 10 vaccine group, Q beta-DK 11 vaccine group, Q beta-DI 10 vaccine group, Q beta-CE 12 vaccine group and amlodipine gavage group, wherein the number of each group is 8. Starting immunization and gastric perfusion from 6 weeks of age, wherein the dose of the gastric perfusion group is 5mg/kg/d, the PBS control group, the VLP group, the Q beta-VC 10 vaccine group, the Q beta-DK 11 vaccine group, the Q beta-DI 10 vaccine group and the Q beta-CE 12 vaccine group take equal volumes of vaccine, Q beta-2 aa and PBS, the dose is 300 ug/mouse, the vaccine is injected into 3-4 points of the subcutaneous part of the back of a rat, and the vaccine is boosted for 3 times every two weeks after primary immunization and is boosted for 1 time. Blood is collected from rat tails at 14,28,42 and 56 days, supernatant is taken after centrifugation at 3000rpm for 10min at normal temperature, the supernatant is diluted at 1:5000, and the titer of antibodies against VC10, DK11, DI10 and CE12 is determined by ELISA method, the trend is shown in figure 2, and the result shows that the antibody against L-type calcium channel immunogenic peptide fragment of vascular smooth muscle cell is generated after rats are immunized by each vector vaccine.

The specific implementation method for detecting the antibody titer by ELISA is as follows:

in order to avoid cross reaction, KLH is used as a carrier to be coupled with the short peptide to form a vaccine, and then the titer of the corresponding antibody is determined by plate wrapping, and the specific process of coupling the KLH as the carrier with the short peptide to form the vaccine is shown in example 2.

1) Wrapping a plate: 100ug of each of the vector vaccines KLH-VC10, KLH-DK11, KLH-DI10 and KLH-CE12 is respectively added into 10ml of coating diluent (PH9.60.05M NaCO3-NaHCO3 buffer solution) to be mixed evenly, then added into a 96-well plate, and incubated at 100 ul/well and a wet box at 4 ℃ overnight;

2) and (3) sealing: discarding the coating solution the next day, adding PBS buffer solution with concentration of 1% BSA, 100 ul/well, blocking for 2h at 37 ℃;

3) sealing the film: sealing, removing the sealing liquid, drying by patting and airing at room temperature, and attaching an ELISA coating film;

4) taking out the serum, thawing the serum on ice, and determining the corresponding antibody titer by an ELISA method, wherein the method comprises the following specific steps:

4a) dilution by multiple times: serum supernatant was diluted with 10% FBS in PBS buffer as a diluent, and a 1: diluting by 5000 times, and adding into a 96-well plate in the step 1);

4b) incubating the primary antibody: placing the 96-well plate in an incubator at 37 ℃ and incubating for 2 h;

4c) incubation of secondary antibody: after the primary antibody incubation is finished, liquid is discarded, washed for 3 times by washing liquid (0.03% PBST PH7.4), dried, and then a goat anti-rat secondary antibody (10% FBS PBS buffer solution is diluted by 1: 3000) marked by horseradish peroxidase is added to incubate for 0.5h in an incubator at 37 ℃;

4d) color development: after the secondary antibody incubation is finished, discarding liquid, washing for 3 times by using a washing solution (0.03% PBST PH7.4), patting dry, then adding TMD developing solution, 100 ul/hole, and observing color change at room temperature;

4e) and (3) terminating the reaction: adding 100ul of stop solution (1M diluted hydrochloric acid) per well after the color of the blank control well begins to turn green;

4f) reading: after adding the stop solution, placing the mixture on an enzyme-linked immunosorbent assay (ELISA) reader and reading the absorbance (OD) value at the wavelength of 450 nm;

4g) and (4) analyzing results: and (4) taking the OD value not less than 2.1 times of that of the blank control group as the positive standard of the sample to be detected, and then calculating the antibody titer value of the corresponding sample.

The blood pressure of the PBS control group, VLP group, Q β -VC10 vaccine group, Q β -DK11 vaccine group, Q β -DI10 vaccine group, Q β -CE12 vaccine group, amlodipine group SHRs was measured every week by tail vein method 8 times, and the average value was taken. Compared with the PBS group, the VLP group, the Q beta-VC 10 vaccine group, the Q beta-DI 10 vaccine group and the DK-11 group have no obvious difference, the blood pressure of SHRs of the Q beta CE-12 vaccine group slightly decreases in one week after immunization compared with the PBS control group, the SHRs reach a peak in one month, the average SHRs decrease by 19.8mmHg, and the blood pressure trend is shown in figure 3.

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Sequence listing

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Claims

1. A human vascular smooth muscle cell L-type calcium channel immunogenic peptide fragment is characterized in that: the amino acid sequence of the L-type calcium channel immunogenic peptide fragment of the human vascular smooth muscle cell is CAPESEPSNSTE.

2. A human vascular smooth muscle cell L-type calcium channel immunogenic carrier vaccine is characterized in that: the human vascular smooth muscle cell L-type calcium channel immunogenic carrier vaccine is prepared by coupling the human vascular smooth muscle cell L-type calcium channel immunogenic peptide CAPESEPSNSTE of claim 1 with a carrier.

3. The human vascular smooth muscle cell L-type calcium channel immunogenic carrier vaccine of claim 2, which is characterized in that: the carrier is Q beta-2 aa phage virus-like particle protein or keyhole limpet hemocyanin.

4. Use of the human vascular smooth muscle cell L-type calcium channel immunogenic carrier vaccine of claim 2 in the preparation of a medicament for treating essential hypertension.