CN1312173C - Section of synthesized peptide S28-42 of troponin I of human cardiac muscle and application - Google Patents

Abstract

The present invention relates to a synthetic peptide section S<28 to 42> used for detecting people's myocardial damage, and an application thereof. The peptide section has strong immunogenicity and immunoreactivity, and can replace people's myocardial troponin I (cTnl) to immunize high titer and high specificity cTnl antibodies generated by various vertebrates. Similar peptide sections remaining in serum after cTnl degradation can be detected by the antibody prepared by the peptide S<28 to 42> immunization. The synthetic peptide section can be used for preparing people's myocardial damage detecting kits.

Description

A kind of human cardiac troponin I section of synthesized peptide S 28-42And uses thereof

Technical field

The present invention relates to a kind ofly can be used for producing and screening at the efficient antiserum(antisera) of a certain special antigenic determinant epitope of human cardiac troponin I and the polypeptide of high degree of specificity monoclonal antibody, and the application of this polypeptide.

Background technology

Myocardial ischemic injury, particularly Acute Myocardial Infarction (AMI) are one of principal diseases that threatens the human life, and people are striving to find the index good, highly sensitive to the AMI specificity always.Along with further investigation to myocardium mark, biochemical marker develops into present multiple early sign thing (as myohaemoglobin etc.) and specificity marker thing (cardiac troponin) from previous aspartate aminotransferase (AST), serum lactic dehydrogenase (SLDH) (LD) and isozyme, creatine kinase (CK) and isozyme, and the susceptibility of diagnosis, specificity more become to strengthening.

Cardiac muscle troponin I (cTnI) is the only specific antigen of cardiac muscle, as the sign of myocardial cell injury, the high degree of specificity in circulation of blood and and susceptibility, be not subjected to people's attention more more.1994～nineteen ninety-five cTnT, cTnI are used for clinical AMI diagnosis by drugs approved by FDA respectively.CTnI has been used for the differential diagnosis of emergency room to pectoralgia, its releasing pattern is similar to CK-MB (pectoralgia 4～6 hours), but cTnI level rising time length (6～10 days) is than the obvious length of CK-MB, and particularly those patients that go to a doctor immediately after the AMI symptom occurring are more not useful.

CTnI one has 209 amino acid whose polypeptide, and relative molecular weight is 24000, and the pI of prediction is about 10.The molecular weight of two kinds of skeletal troponins (ssTnI and fsTnI) is about 20KD.Utilize computer program that the cTnI secondary protein structure is carried out prediction result and show that cTnI is a kind of protein that contains 5 a spirals.Conjugated antigen epi-position experimental result can infer that cTnI is not a sphaeroprotein, and its protein peptide chain may be taked a kind of conception of more stretching, the protein molecule of this structure, and its most of sequence all has antigenicity, can be by immune system recognition.The immunogenicity of cTnI whole protein is very weak, has increased the difficulty of utilizing cTnI protein Preparation polyclone and monoclonal antibody, and the monoclonal antibody of utilizing whole protein to prepare can be at a lot of epi-positions.The complicacy of the own biochemical property of cTnI and in the existence form of patient's peripheral blood makes to exist difference between the measured value of cTnI detection reagent, surplus different cTnI analytical procedures can differ 20 to the detected value maximum with a sample times.U.S. clinical pathology association (CAP) was investigated Access, OpusPlus and three systems of Stratus of detecting cTnI in 1997, found that average cTnI concentration is owing to the analytical system difference of using has substantial difference.The measured value of Access is minimum, some in addition lower 20 times than OpusPlus result.Causing one of reason that same duplicate samples is measured difference is the different determinant of antibody recognition that provides in the test kit, and another prior factor is exactly that standard antigen in the test kit exists very big difference.The N-terminal part of Stratus and Opus antibody recognition cTnI, and the C-terminal part of the antibody recognition cTnI of ACCESS.Application is to N-terminal part and the special monoclonal antibody of C-terminal part of cTnI, to normal human serum that is added with reorganization cTnI and the normal human serum that is added with reorganization cTnI mixture, 37 ℃ hatch 2,4,6,24 and 48 hours respectively after, the result who carries out westerm blot analysis shows, easier being degraded of C-terminal part of cTnI.This also may be to cause the one of the main reasons that makes a variation between these three kinds of measuring methods.U.S. clinical chemistry association has researched and analysed 10 kinds of cTnI standard substance recently, but they are all unsatisfactory.This makes the term of reference of the whole bag of tricks, the threshold value etc. of analyzing interference, analytical variance, diagnostic window phase and diagnosis and prognosis difference in various degree occur, brings certain puzzlement for clinical application and evaluation.When commodity in use cTnI detection kit, must study the characteristic of commercially available reagent.So it is a very major issue that cTnI lacks bioassay standardization.The most of sequence of cTnI protein all has antigenicity, and wherein the antigenicity of N-terminal and C-terminal is the strongest.It is 40% different that cTnI and the aminoacid sequence of sTnI have approximately.32 amino-acid residues are arranged is that skeletal troponin (sTnI) does not have to people cTnI N-terminal in addition, so antigenicity has than big-difference, helps obtaining specificity cTnI antibody.

CTnI is very unstable in serum, easily by proteasome degradation, in patient AMI, exist with the cTnl-TnC composite form more than the 90%cTnI, and N-holds and the C-end then is subject to followed by action of proteolytic enzymes to the followed by action of proteolytic enzymes sensitivity, finds degraded rapidly in experiment in vitro.Though people cTnl N-terminal is the strongest epitope district with completing basic end, this zone is also comparatively responsive to oxidation, reduction and phosphorylation, and also easily by the hydrolysis of endogenous protein lytic enzyme, different states possesses different immunocompetences.Because the protection of TnC, stabilized zone is between 28～110 amino-acid residues, and the antibody that can discern this section stable region can improve susceptibility and the repeatability that cTnI detects, and helps to improve the stdn that cTnI detects.

Summary of the invention

The object of the present invention is to provide a kind of susceptibility of the cTnI of raising detection and the section of synthesized peptide of the cardiac troponin of repeatability.

Another object of the present invention is the monoclonal antibody hybridoma cell strain of screening at this peptide section, is used to prepare the cardiac troponin detection kit.

The object of the present invention is achieved like this:

1. the selection of peptide sequence:

Utilize the DNA-Star analysis software to its antigenicity of cTnI sequential analysis of protein, hydrophobicity, snappiness, surperficial accessibility, again in conjunction with proteic biochemical property of cTnI and the existence in serum thereof, and the difference of human cardiac troponin I (cTnI) and skeletal troponin (ssTnI and fsTnI) sequence, the difference of the cardiac muscle troponin I sequence of human cardiac troponin I and mouse, we have selected one section aminoacid sequence of 28-42AA, are designated as SEQ NO.1.Sequence is as follows:

AYATEPHAKKKSKIS

2. polypeptide synthesis method:

Consider the stability of polypeptide, the present invention makes N-end Ala acetylize when synthetic, add a Cys behind C-end Ser, is designated as S28-42.Sequence is as follows:

CAYATEPHAKKKSKIS-NH2

This sequence has following application:

1) substitutes the natural cardiac muscle troponin I of people as immunogen, and have the immunogenicity higher, help efficient sero-fast generation at the cTnI antigenic determinant than cardiac muscle troponin I holoantigen.

2) help generation at the monoclonal antibody specific cell strain of cTnI antigenic determinant.

3) be used to screen cell strain of monoclonal antibody at cTnI.

4) with polypeptide S _28-42Carry out chemical commissure or gene engineering expression with other protein and as antigen, carry out immune animal with preparation antibody and as the contrast of detection cTnI.

Special peptides sequence S provided by the invention _28-42Antigenic determinant structural similitude with natural human cardiac troponin I has strong immunogenicity and immunoreactivity, with this peptide S _28-42The antibody of immunity preparation remains in peptide section similar in the serum after can detecting the cTnI degraded, with the reagent of this peptide section preparation to cTnI that myocardial damage was discharged detect higher level, repeatability is better, and this peptide section can replace cTnI albumen as antigen, to avoid the cTnI whole protein as immunogenic weak immunogenicity.

Embodiment

1. adopt synthetic peptide S _28-42With the MBS of carrier protein couplet (-maleimide phenylformic acid-N-hydroxy-succinamide fat) method, because S _28-42With people's the skeletal troponin and the difference of mouse cardiac muscle troponin sequence, obtain higher immune effect after the immune BALB/c mouse.Concrete grammar is as follows:

1) between-maleimide phenylformic acid-N-hydroxy-succinamide fat (MBS) is mixed with the dimethyl formamide (DMF) of 5mg/ml, with preparation in last hour.

2) keyhole relative hemocyanin (KLH) 10mg is dissolved in the 1ml deionized water.

3) 1ml KLH solution is put into small test tube, adds the DMF solution 140ul that is dissolved with 2mg MBS.Stirring at room 30min.(PBS PH7.4), collects gray product to cross the G-25 post.The 5mg polypeptide is dissolved among the PBS (0.01mol/L) of the PH7.4 that is not more than 0.5ml.The two mixing, stirring at room 2 hours.4 ℃ of dialysed overnight, packing-20 ℃ preservation.With this synthetic peptide S _28-42The immunity BALB/c mouse, ELISA detects, and the mouse resisting anteserum that obtains is tired up to 10 ⁶

2. because S _28-42With the difference of people's skeletal troponin sequence, use S _28-42The monoclonal antibody at this section sequence that screening obtains can be avoided the cross reaction with skeletal troponin significantly in reagent detects.

Use S _28-42The monoclonal antibody at this section sequence that screening obtains detects cardiac muscle troponin I and skeletal troponin respectively by the ELISA method, and cross reacting rate is less than 0.01%.Explanation is by S _28-42The monoclonal antibody that screening obtains can be used to prepare the cardiac troponin detection kit, has using value in the exploitation of detection kit.

3. use S _28-42The monoclonal antibody specific that obtains of screening can be discerned the section of cardiac muscle troponin I stable existence among the patients serum, can help to analyze patient fall ill stage and degree.

Claims (2)

1. section of synthesized peptide S _28-42, it is characterized in that being selected from the amino acid of the 28-42 of human cardiac troponin I, its aminoacid sequence is: CAYATEPHAKKKSKIS-NH ₂

2. by the described section of synthesized peptide S of claim 1 _28-42Application, it is characterized in that: be used to screen monoclonal antibody hybridoma cell strain, and this monoclonal antibody is used to prepare the cardiac troponin detection kit at above-mentioned peptide section sequence.