CN103694355B - The recombinant antibodies of anti-human cardiac muscle troponin I and construction method thereof and application - Google Patents
The recombinant antibodies of anti-human cardiac muscle troponin I and construction method thereof and application Download PDFInfo
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Abstract
The present invention relates to the recombinant antibodies of a kind of anti-human cardiac muscle troponin I, it includes the light chain being made up of the variable region of light chain of mouse source antibody of anti-human cardiac muscle troponin I and the constant region of light chain of human IgG1 and the heavy chain being made up of the variable region of heavy chain of mouse source antibody of anti-human cardiac muscle troponin I and the CH of human IgG1.This recombinant antibodies is compared with murine antibody, both remained parental antibody identifies the specific of antigen and affinity, and avoid and the anti-mouse antibody generation nonspecific reaction in human serum, therefore, from the point of view of in terms of clinical diagnostic applications, above-mentioned recombinant antibodies has more using value than murine antibody.Moreover, it relates to the construction method of the recombinant antibodies of a kind of anti-human cardiac muscle troponin I and the application of this recombinant antibodies.
Description
Technical field
The present invention relates to antibody engineering field, especially relate to the recombinant antibodies of a kind of anti-human cardiac muscle troponin I
And construction method and application.
Background technology
Before the eighties in 20th century, the World Health Organization (WHO) always using myocardial enzymes activity as Acute myocardial
One of diagnostic criteria of infarct (AMI).Late 1980s, scientific research personnel finds, troponin
The Sensitivity and Specificity of (troponin, Tn) is higher than creatine phosphokinase (CK), CPK-MB
(CK-MB), the biomarker such as lactic dehydrogenase and aspartate aminotransferase.Cardiac muscle troponin I
(cTnI) existing only in cardiac muscle, be the mark of cardiac muscle cell, its abnormal change can affect the systolic and diastolic function of heart,
And can be used for diagnosing myocardial necrosis, it is judged that myocardial damage etc., become myocardial cell injury Sensitivity and Specificity
One of the strongest mark, quick diagnosis AMI being well recognized as and acute coronary syndrome (acute coronary
Syndromes, ACS) and assist ACS risk stratification and reflect the main biochemical mark of its prognosis.
In normal human blood, cTnI content is generally below 0.3 μ g/L.Cardiac muscle cells after birth integrality because of ischemic or
When anoxics etc. are destroyed, free cTnI can enter blood flow through cell membrane rapidly.Therefore, at the beginning of morbidity
Phase quick, sensitive and measure accurately the cTnI in human blood and variation tendency thereof to the diagnosis of acute myocardial infarction AMI,
The myocardial damage etc. that the risk stratification of acute coronary syndrome, monitoring various factors cause has important
Clinical meaning.The method being used for detecting cTnI level clinically has enzyme linked immunosorbent assay (ELISA), chemistry
Luminescence, collaurum etc., distinct methods has respective pluses and minuses, but is required for being directed to the special of cTnI
Property monoclonal antibody.What traditional clinical diagnosis used is all the monoclonal antibody (mAb) in mouse source.
For a long time, the monoclonal antibody of mouse is widely used in scientific research, clinical diagnosis and treatment,
But owing to the mAb of mouse can cause HAMA (HAMA) to react, thus limit it and facing
Application in terms of Chuan.Therefore, murine antibody is carried out humanization modified being very important.From last generation
Having recorded since the eighties, the development trend for the monoclonal antibody for the treatment of is to humanized antibody from mouse source antibody
Arriving human antibody again, the most having in the antibody drug by FDA approval listing is closely partly humanized antibody.
And in clinical diagnosis field, the antibody of the substantially still mouse currently used, but owing to people usually can
Touch the article that muroid contacted, it is possible that HAMA reacts in blood, thus there is the anti-mouse of people
Antibody, there will be nonspecific reaction in clinical diagnosis, i.e. there will be false positive.
Summary of the invention
Based on this, it is necessary to provide a kind of anti-human cardiac muscle troponin I that can significantly reduce false positive probability of occurrence
Recombinant antibodies and construction method and application.
The recombinant antibodies of a kind of anti-human cardiac muscle troponin I, resists including by the mouse source of anti-human cardiac muscle troponin I
Light chain that the constant region of light chain of the variable region of light chain of body and human IgG1 is constituted and by anti-human cardiac muscle troponin I
The heavy chain that the CH of the variable region of heavy chain of mouse source antibody and human IgG1 is constituted.
Wherein in an embodiment, the mouse source antibody of described anti-human cardiac muscle troponin I by preserving number is
The hybridoma of CCTCC C2013185 produces.
Wherein in an embodiment, described variable region of light chain contains the amino acid as shown in SEQ ID NO.1
Sequence.
Wherein in an embodiment, described variable region of heavy chain contains the amino acid as shown in SEQ ID NO.2
Sequence.
The construction method of a kind of anti-human cardiac muscle troponin I recombinant antibodies, comprises the steps:
Build the expression containing the nucleotide sequence as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively to carry
Body, wherein, the expression vector containing the nucleotide sequence shown in SEQ ID NO.3 contains the light chain of human IgG1
The expressing gene of constant region, the expression vector containing the nucleotide sequence shown in SEQ ID NO.4 contains people
The expressing gene of the CH of IgG1；
Described expression vector is transfected in same host cell；
Reclaim from described host cell and obtain described anti-human cardiac muscle troponin I recombinant antibodies.
Wherein in an embodiment, the expression being inserted into the nucleotide sequence as shown in SEQ ID NO.3 carries
Body is reserved with XbaI and PmlI double enzyme site；It is inserted into the nucleotide sequence as shown in SEQ ID NO.4
Expression vector be reserved with NheI and HindIII double enzyme site.
Wherein in an embodiment, described nucleotide sequence as shown in SEQ ID NO.3 is by as follows
Step prepares:
From the hybridoma that preserving number is CCTCC C2013185, extract RNA, use Reverse Transcription box
Carry out after 70 DEG C of enzymes of RT-PCR, RT-PCR amplified production inactivate as template, with SEQ ID NO.5 and SEQ
Primer sequence shown in ID NO.6 carries out PCR, reclaims PCR primer, and PCR primer rTaq DNA gathers
Synthase is inserted in pMD-18T carrier after carrying out adding A reaction, is transformed in DH5 α competent cell expression；
Reclaim the product that DH5 α competent cell is expressed, use such as SEQ ID NO.7 and SEQ ID NO.8
Shown primer sequence carries out PCR, obtains the nucleotide sequence shown in described SEQ ID NO.3；
Described nucleotide sequence as shown in SEQ ID NO.4 is to be made by the steps acquisition:
From the hybridoma that preserving number is CCTCC C2013185, extract RNA, use Reverse Transcription box
Carry out after 70 DEG C of enzymes of RT-PCR, RT-PCR amplified production inactivate as template, with SEQ ID NO.9 and SEQ
Primer sequence shown in ID NO.10 carries out PCR, reclaims PCR primer, and PCR primer rTaq DNA gathers
Synthase is inserted in pMD-18T carrier after carrying out adding A reaction, is transformed in DH5 α competent cell expression；
Reclaim the product that DH5 α competent cell is expressed, use such as SEQ ID NO.11 and SEQ ID NO.12
Shown primer sequence carries out PCR, obtains the nucleotide sequence shown in described SEQ ID NO.4.
A kind of DNA of coding amino acid sequence as shown in SEQ ID NO.1.
A kind of DNA of coding amino acid sequence as shown in SEQ ID NO.2.
A kind of DNA comprising coding amino acid sequence as shown in SEQ ID NO.1 and/or coding are such as SEQ
The eukaryotic expression vector of the DNA of the amino acid sequence shown in ID NO.2.
A kind of host cell transfected by above-mentioned eukaryotic expression vector.
The application in preparation detection cardiac muscle troponin I detection reagent or detecting instrument of the above-mentioned recombinant antibodies.
The Test paper of a kind of cardiac muscle troponin I, including above-mentioned recombinant antibodies.
Wherein in an embodiment, described Test paper includes support slice, sample pad, gold mark pad, nitre
Acid cellulose film, absorption pad, detection line and nature controlling line, described sample pad, described gold mark pad, described nitric acid
It is thin that cellulose membrane and described absorption pad are successively set on described support from one end of described support slice to the other end
On sheet, described sample pad partly overlaps with described gold mark pad, described gold mark pad and described nitrocellulose filter portion
Dividing overlap, described nitrocellulose filter partly overlaps with described absorption pad, described detection line and described nature controlling line
It is located on described nitrocellulose filter, and described detection line is located at the one end near described gold mark pad, described matter
Control line is located at the one end near described absorption pad, and described gold mark pad is coated with the described attached colloid of recombinant antibodies bag
Gold grain forms the recombinant antibodies of colloid gold label, and described detection line is the anti-cardiac muscle troponin I of affinity purification
Rabbit resist more, described nature controlling line is sheep anti-mouse igg antibody.
A kind of detection kit, including the Test paper of above-mentioned cardiac muscle troponin I.
Above-mentioned recombinant antibodies is compared with murine antibody, and both remained parental antibody identifies the specific of antigen
And affinity, and avoid and the anti-mouse antibody generation nonspecific reaction in human serum, therefore, from facing
From the point of view of bed diagnostic application aspect, above-mentioned recombinant antibodies has more using value than murine antibody.
Accompanying drawing explanation
Fig. 1 is the front schematic view of the Test paper of an embodiment；
Fig. 2 is the Longitudinal cross section schematic of Test paper in Fig. 1；
Fig. 3 is detection kit schematic diagram；
Fig. 4 be the cDNA that obtains after the RNA reverse transcription that hybridoma extracts as template, with heavy chain and
The amplification of light chain degenerate primer is containing VHAnd VLThe electrophoretogram of genetic fragment, VLMKF5/mKR primer to
VHMHF2/mHR primer pair amplifies go out the band of about 420bp, M is DL2000DNAMarker；
Fig. 5 is recombinant antibodies carrier for expression of eukaryon, a be pFP-IgCK, b be pFP-IgCH；
Fig. 6 is for using VLAnd VHThe V of special primer amplificationL(395bp) and VH(405bp) gene sheet
Section, M is DL2000DNAMarker；
Fig. 7 be recombinant antibodies proteinG affinity column purify time penetrate peak and absworption peak；
Fig. 8 analyzes for SDS-PAGE after restructuring antibody purification, and a is 4 μ g mouse source antibody, and b is 4 μ g restructuring
Antibody, M is Protein Marker.
Detailed description of the invention
Below in conjunction with drawings and the specific embodiments to the recombinant antibodies of the anti-human cardiac muscle troponin I of the present invention and
Its construction method and application are described in further detail.
The recombinant antibodies of the anti-human cardiac muscle troponin I of one embodiment, including by anti-human cardiac muscle troponin I
(hcTnI) variable region of light chain (L chain V district, the V in mouse source antibody (mAb)L) light with human IgG1
Chain constant region (L chain C district, CL, such as k chain C district (Ck) etc.) light chain that constitutes and by anti-human myocardium myo
Variable region of heavy chain (H chain V district, the V of the mouse source antibody of calcium protein IH) and the CH (H of human IgG1
Chain C district, CH) heavy chain that constitutes.
Wherein, the mouse source antibody of above-mentioned anti-human cardiac muscle troponin I is CCTCC C2013185 by preserving number
Hybridoma produce, the named CTNI-C4 of this hybridoma, on November 13rd, 2013 protect
Ensconce Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center (i.e. in China typical culture collection
The heart).Above-mentioned variable region of light chain contains the amino acid sequence as shown in SEQ ID NO.1, the DNA of its correspondence
Sequence is SEQ ID NO.3 in sequence table；Variable region of heavy chain contains the amino acid as shown in SEQ ID NO.2
Sequence, the DNA sequence dna of its correspondence is SEQ ID NO.4 in sequence table.
The sequence of SEQ ID NO.3 is: GAT GTTTTGATGA CCCAAACTCC ACTCTCCCTG CCTGTCAGTC TTGGAGATCA GCCTCCATC TCTTGC TGGTAC CTGCAGAAAC CAGGCCAGTC TCCAAAACTC CTGATCTAC GGGGTCCCAG ACAGGTTCAG TGGCAGTGGA TCAGGGACAG ATTTCACACT CAAGATCAGC AGAGTGGAGG CTGAGGATCT GGGAGTTTAT TACTGC TTCGGTG CTGGGACCAA GCTGGAGCTG AAACGG.The sequence of SEQ ID NO.4
It is classified as: CAG GTCCAACTGC AGCAGCCTGG GTCTGAACTG GTGAGGCCTG GGGCTTCAGT GAAGCTGTCC TGCAAGGCTT CTGGCTACAC CTTCACC TGGGTGAA GCAGAGGCCT GGACAAGGCC TTGAATGGAT TGGT AAGGC CACATTGACT GTAGACAAAT CGTCCAGCGC AGCCTACATG CACCTCAACA GCCTGACATC TGAGGACTCT GCGGTCTATT ACTGTGCACA A TGGGGTCA AGGAACCTCA GTCACTGTCT CTGCA.Wherein, with a underscore mark
Remember is signal peptide (Signal Peptide, the SP) sequence of antibody, is frame by the sequence of real underscore mark
Frame district (framework Region, FR) sequence, sequences in italics is complementary determining region (complementary
Determining region, CDR) sequence, i.e. hypervariable region.
Present embodiment additionally provides the construction method of a kind of anti-human cardiac muscle troponin I recombinant antibodies, including such as
Lower step:
Build the expression containing the nucleotide sequence as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively to carry
Body, wherein, the expression vector containing the nucleotide sequence shown in SEQ ID NO.3 contains the light chain of human IgG1
The expressing gene of constant region, the expression vector containing the nucleotide sequence shown in SEQ ID NO.4 contains people
The expressing gene of the CH of IgG1；
Expression vector is transfected in same host cell；
Reclaim from host cell and obtain anti-human cardiac muscle troponin I recombinant antibodies.
Wherein, be inserted into the expression vector of the nucleotide sequence as shown in SEQ ID NO.3 be reserved with XbaI and
PmlI double enzyme site；The expression vector being inserted into the nucleotide sequence as shown in SEQ ID NO.4 is reserved with
NheI and HindIII double enzyme site.
Nucleotide sequence as shown in SEQ ID NO.3 is to be made by the steps acquisition:
From the hybridoma that preserving number is CCTCC C2013185, extract RNA, use Reverse Transcription box
Carry out after 70 DEG C of enzymes of RT-PCR, RT-PCR amplified production inactivate as template, with SEQ ID NO.5 and SEQ
Primer sequence shown in ID NO.6 carries out PCR, reclaims PCR primer, and PCR primer rTaq DNA gathers
Synthase is inserted in pMD-18T carrier after carrying out adding A reaction, is transformed in DH5 α competent cell expression；
Reclaim DH5 α competent cell to express
Product, use primer sequence as shown in SEQ ID NO.7 and SEQ ID NO.8 to carry out PCR,
Obtain the nucleotide sequence shown in SEQ ID NO.3.
Nucleotide sequence as shown in SEQ ID NO.4 is to be made by the steps acquisition:
From the hybridoma that preserving number is CCTCC C2013185, extract RNA, use Reverse Transcription box
Carry out after 70 DEG C of enzymes of RT-PCR, RT-PCR amplified production inactivate as template, with SEQ ID NO.9 and SEQ
Primer sequence shown in ID NO.10 carries out PCR, reclaims PCR primer, and PCR primer rTaq DNA gathers
Synthase is inserted in pMD-18T carrier after carrying out adding A reaction, is transformed in DH5 α competent cell expression；
Reclaim the product that DH5 α competent cell is expressed, use such as SEQ ID NO.11 and SEQ ID NO.12
Shown primer sequence carries out PCR, obtains the nucleotide sequence shown in SEQ ID NO.4.
Present embodiment applies the primer of an Analysis of Nested Design to resist from the anti-hcTnI mouse monoclonal independently building and cultivating
Body hybridoma cell strain (CCTCC C2013185) has cloned the light chain of anti-hcTnI antibody and heavy chain
Variable region gene.Light chain after order-checking and weight chain variabl area sequence are carried out point in IMGT antibody database
Analysis, result shows that VH gene (heavy chain variable region gene) has higher with the mouse source VH gene in database
Homology, and belong to mouse VH1 gene family；In VL gene (chain variable region gene) and database
Mouse source VL gene there is higher homology, and belong to mouse Vk2 gene family.
Additionally, present embodiment additionally provides one comprises coding SEQ ID NO.3 and/or SEQ ID NO.4
The expression vector of the DNA of sequence, as conventional in pFP-IgCH and pFP-IgCK or genetic engineering other are true
Nuclear expression carrier, and the host cell of this DNA expression vector transfection, such as Freestyle CHO-S cell
Or other expression cells that genetic engineering is conventional.
Above-mentioned recombinant antibodies can be widely used in preparation detection cardiac muscle troponin I detection reagent or detecting instrument
Field in.Such as, the detection kit of a kind of cardiac muscle troponin I, it include housing, Test paper and its
He detects reagent.
As depicted in figs. 1 and 2, the Test paper 100 of present embodiment includes support slice 110, sample pad
120, gold mark pad 130, nitrocellulose filter 140, absorption pad 150, detection line 160 and nature controlling line 170.
Sample pad 120, gold mark pad 130, nitrocellulose filter 140 and absorption pad 150 are from the one of support slice 110
End is successively set on support slice 110 to the other end.Sample pad 120 partly overlaps with gold mark pad 130,
Gold mark pad 130 partly overlaps with nitrocellulose filter 140, nitrocellulose filter 140 and absorption pad 150
Divide overlap.Detection line 160 and nature controlling line 170 are located on nitrocellulose filter, and detection line 160 is located at and leans on
One end of nearly gold mark pad 130, nature controlling line 170 is located at the one end near absorption pad 150.Support slice 110
The material not absorbed water is used to make.Sample pad 120 is for sample point sample.The attached colloid gold particle of recombinant antibodies bag
The recombinant antibodies forming colloid gold label is coated uniformly on gold mark pad 130.Detection line 160 is affinity purification
The rabbit of anti-cardiac muscle troponin I resist more, nature controlling line 170 is sheep anti-mouse igg antibody.
As it is shown on figure 3, Test paper 100 is placed in the housing 200 of detection kit.On housing 200
Offer well 210 and observation window 220.The position of well 210 counter sample pad 120.Detection line
160 and nature controlling line 170 be exposed in observation window 220, convenient observe.
Other detection reagent can directly be prepared in laboratory as required.
Above-mentioned detection kit utilizes double antibody sandwich method to detect the cardiac muscle troponin I in tested material.Inspection
During survey, in sample, all of cTnI is first and gold marks the recombinant antibodies combination of anti-cardiac muscle troponin I, due to
Capillarity, reaction compound, along nitrocellulose filter 140 swimming forward, if there being cTnI in sample, arrives
When reaching detection line 160, the rabbit running into the anti-cardiac muscle troponin I being coated on nitrocellulose filter 140 resists more,
The how anti-cardiac muscle troponin I of rabbit-gold mark recombinant antibodies compound will be formed, thus be enriched in detection line 160
On, form red precipitate line；The gold mark recombinant antibodies of uncombined cTnI is then by detection line 160, by sheep
Dynamics captures, and is enriched on nature controlling line 170, forms red precipitate line.When detection line 160 with
It is judged to positive findings when having red precipitate line on nature controlling line 170 simultaneously.If sample does not contains cTnI, reaction
Compound arrives when detecting line 160, runs into the many anti-rabbit how anti-cTnI-gold marks that would not be formed of capture and recombinates anti-
Nanocrystal composition, reaction compound, by detection line 160, is only enriched on nature controlling line 170 formation red precipitate line,
Now it is judged to negative findings.
Additionally, in other embodiments, the structure of this detection kit is not limited to described above.Above-mentioned heavy
Group antibody is except applying in addition to the monoclonal antibody detection kit of above-mentioned colloid gold label, it is also possible to for other myocardium myos
In calcium protein I detection kit or equipment.It will be understood by those skilled in the art that the restructuring of present embodiment
Antibody directly or indirectly combines other signal group (such as magnetic microsphere, HRPO etc.), or by this reality
Execute the recombinant antibodies of mode as coated antibody (such as ELISA), then can be used for the myocardium myo calcium of other forms
Protein I detection reagent or equipment.
Above-mentioned recombinant antibodies is compared with murine antibody, and both remained parental antibody identifies the specific of antigen
And affinity, and avoid and the anti-mouse antibody generation nonspecific reaction in human serum, therefore, from facing
From the point of view of bed diagnostic application aspect, above-mentioned recombinant antibodies has more using value than murine antibody.
It is below specific embodiment part:
Freestyle CHO-S cell, transfection reagent FreeStyle in the present embodimentTMMAX Reagent and thin
Born of the same parents' culture medium etc. is purchased from Life Technologies company.Prime Star archaeal dna polymerase is purchased from Takara
Company.TrizolRNA extracts kit purchased from Sangon Biotech (Shanghai) Co., Ltd..In restricted
Cut enzyme purchased from NEB company.Plasmid extraction kit is purchased from Tian Gen company.Primer synthesis and gene sequencing by
Invitrogen company completes.People's cardiac muscle troponin I antigen of recombinating is Shenzhen's phenanthrene roc limited public affairs of biology share
Department produces, article No. AG-CTNI-HP0005.
1, design of primers and synthesis
Amplification VLUpstream region of gene primer:
MkF1:5 '>ATGGAGACAGACACACTCCTGCTAT<3 ' (SEQ ID NO.13)；
MkF2:5 '>ATGGATTTTCAAGTGCAGATTTTCAG<3 ' (SEQ ID NO.14)；
MkF3:5 '>ATGGAGWCACAKWCTCAGGTCTTTRTA<3 ' (SEQ ID NO.15)；
MkF4:5 '>ATGKCCCCWRCTCAGYTYCTKGT<3 ' (SEQ ID NO.16)；
MkF5:5 '>ATGAAGTTGCCTGTTAGGCTGTTG<3 ' (SEQ ID NO.5).
Amplification VLDownstream of gene primer:
MkR:5 '>GGATACAGTTGGTGCAGCATCAGCCCGTTT<3 ' (SEQ ID
NO.6).
Amplification VHUpstream region of gene primer:
MHF1:5 '>SAGGTGMAGCTKCASSARTCWGG<3 ' (SEQ ID NO.17)；
MHF2:5 '>ATGGRATGSAGCTGKGTMATSCTCT<3 ' (SEQ ID NO.9)；
MHF3:5 '>ATGRACTTCGGGYTGAGCTKGGTTTT<3 ' (SEQ ID NO.18)；
MHF4:5 '>ATGGCTGTCTTGGGGCTGCTCTTCT<3 ' (SEQ ID NO.19).
Amplification VHThe downstream primer of gene:
MHR:5 '>TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA<3 ' (SEQ ID
NO.10).
2, antibody variable gene clone and order-checking
RNA in extracting from hybridoma cTnI-C4, carries out RT-PCR with Reverse Transcription box, amplification
As template after 70 DEG C of enzyme inactivations of product, carry out PCR, V with the primer of above-mentioned synthesisLGene magnification 5 is managed,
VHGene magnification 4 is managed, wherein VLMKF5/mKR primer pair amplifies go out the purpose band of about 420bp,
VHMHF2/mHR primer pair amplifies go out the purpose band of about 420bp, as shown in Figure 4.Use agarose
Gel purified recovery, product rTaq archaeal dna polymerase is inserted into pMD-18T after carrying out adding A reaction
In carrier, it is transformed in DH5 α competent cell, after growing bacterium colony, takes V respectivelyHAnd VLGene clone each 2
Individual clone send Invitrogen company to check order.
3, the sequence analysis of cTnI-C4 antibody variable gene
The gene order that above-mentioned order-checking obtains is placed in IMGT antibody database and is analyzed, and utilize
VNTI11.5 software is analyzed determining that heavy chain and the light chain primer gene to amplifying is all correct, wherein
The V of the 420bp that mKF5/mKR amplifiesLIn genetic fragment, VLGene order is 339bp, belongs to VkII
Gene family, there is the leader peptide sequences of 57bp in its front；The 423bp's that mHF2/mHR primer pair amplifies goes out
VHIn genetic fragment, VHGene order is 348bp, belongs to VH1 gene family, and its front has 57bp's
Leader peptide sequences.
4, the structure of recombinant antibodies expression plasmid
PFP-IgCK and pFP-IgCH carrier is the recombinant antibodies carrier for expression of eukaryon built, and the former has inserted
Enter the constant region gene of human IgG k chain, and reserved XbaI and PmlI restriction enzyme site；The latter has inserted human IgG1
Weight chain constant area gene, and reserved NheI and HindIII restriction enzyme site, plasmid map is as shown in Figure 5.
According to antibody variable gene sequencing result in above-mentioned pMD-18T, the V of design cTnI-C4 antibodyL
And VHGene-specific primer, two ends are with restriction enzyme site and protection base, and primer is as follows:
C4-VHF:5 ' > GGGGCTAGCATGGAATGGAGCTGTGTCATC < 3 ' (SEQ ID
NO.7)；
C4-VHR:5 ' > CCCAAGCTTGCTGCAGAGACAGTGACTGAGG < 3 ' (SEQ ID
NO.8)；
C4-VKF:5 ' > CTAGTCTAGAATGAAGTTGCCTGTTAGG < 3 ' (SEQ ID
NO.11)；
C4-VKR:5 '>CCGTTTCAGCTCCAGCTTGG<3 ' (SEQ ID NO.12).
The part of underscore is restriction enzyme site, VHSite, two ends is NheI/HindIII, VLGene 5 ' section is
XbaI, 3 ' sections are PmlI, and this enzyme is flush end enzyme, so can not set restriction enzyme site, Prime Star on primer
The fragment of archaeal dna polymerase amplification is flush end.
V through the 396bp that PCR amplifiesLGenetic fragment and the V of 405bpHGenetic fragment, such as Fig. 6
Shown in.VLGene XbaI enzyme cutting, pFP-IgCK XbaI/PmlI double digestion, VHGene and pFP-IgCH
All use NheI/HindIII double digestion, V after fragment and vector purification being reclaimedLGene is connected to pFP-IgCK
In carrier, VHGene is connected in pFP-IgCH carrier, respectively obtains the recombinant expressed matter of heavy chain and light chain
Grain.
5, recombinant antibodies expression plasmid transfection CHO cell, product detects
Day before transfection inoculation 5 × 105/ mL cell is in 6 well culture plates, with containing 8mM glutamine
Freestyle CHO Expression Medium, and 37 DEG C, 8%CO2Incubator in 150rpm circumference shake
Swinging cultivation 16 22h, during transfection, cell density is 1 × 106/ mL, transfection 3mL cell needs plasmid 3.75 μ g
(heavy chain and each 1.875 μ g of light chain expression plasmid), transfection reagent FreeStyleTMMAX Reagent needs
3.75 μ L, the transfection method recommended according to Life Technologies transfects, and can sample after transfection 72h
The recombinant antibodies that detection is expressed.
6, the recombinant antibodies detection of eukaryotic expression
With 0.06M pH9.6 carbonate buffer solution, dilution people recombinates cardiac muscle troponin I antigen (Shenzhen phenanthrene roc
Biological Co., Ltd. produces, article No. AG-CTNI-HP0005) so that it is final concentration of 8 μ g/mL.Add
Entering 96 hole polystyrene plates, every hole 0.1mL, 37 DEG C of 2 hours or 4 DEG C are overnight.Next day, with little containing 10%
0.02M pH7.2PB, the 0.15mL/ hole of cow's serum (NBS), closes 2 hours, is used for detecting for 37 DEG C.
After transfection the 6th day, take cell conditioned medium 0.1mL in above-mentioned 96 holes detection plates, 37 DEG C 30 minutes, wash six
Mouse-anti human IgG-18#(Shenzhen phenanthrene the roc of the HRPO mark adding 2000 times of dilutions after secondary is biological
Limited company produces), after 37 DEG C are ibid washed for 30 minutes, every hole adds 100 μ L containing 0.1%(M/V)
O-phenylenediamine, 0.1%(V/V) hydrogen peroxide, pH5.0 citrate phosphate buffer, 37 DEG C 15 minutes, add
Enter dilution heat of sulfuric acid, every hole 50 μ L, survey 450nm absorption value.Empty carrier pFP-IgCk and pFP-IgCH is altogether
The cell conditioned medium of transfection, as negative control, must be judged to the positive than 2.0 with measured value with control value.Result
Such as table 1:
Table 1
Result shows and successfully gives expression to activated recombinant antibodies.
7, recombinant antibodies purifies
Amplifying by same method and express 500mL, after expressing six days, cell culture fluid 12000rpm is centrifuged
20min, supernatant transfers to, in clean bottle, carry out affinity purification with proteinG affinity column, purifying
Penetrate peak and absworption peak as shown in Figure 7.Obtain 15mg restructuring cTnI-C4 antibody after purification, take 4 μ g and purify
Antibody carry out reproducibility SDS-PAGE, 4 μ g mouse source cTnI-C4 antibody as comparison, electrophoretogram such as Fig. 8
Shown in.After reproducibility SDS-PAGE, show that two bands, 1 Mr are 50KD(heavy chain), another
Mr is 28KD(light chain).
8, restructuring cTnI-C4 antibody is for cardiac muscle troponin I gold-immunochromatographyreagent reagent for assay box
This detection kit includes Test paper and sample diluting liquid.Wherein, sample diluting liquid is 8%NaCl
Solution.Compound method: 80gNaCl, adds distilled water and is settled to 1000mL.
This Test paper makes as follows:
A. the preparation of nitrocellulose filter
It is coated the preparation of buffer solution: containing 6% methyl alcohol, 0.01M pH7.2PBS buffer solution for being coated buffer solution,
0.22 μm membrane filtration mistake, put 4 DEG C standby, the term of validity one week.The 0.01M pH7.2PBS of 1000mL6% methyl alcohol
Buffer formulation: NaCl8g, KCl0.2g, Na2HPO4·12H2O2.9g、KH2PO40.2g, methyl alcohol 60mL,
Double distilled deionized water is settled to 1000mL.
The preparation of nitrocellulose filter: anti-cardiac muscle troponin I rabbit polyclonal antibody is (deep with being coated buffer solution
Fei Peng Biological Co., Ltd. of ditch between fields city produces, article No. PAB-CTNI-AP0002) it is diluted to 1～5mg/mL,
Adjusting machine, be scribed ss T line, be detection line, T line is near gold mark pad end, away from gold mark pad end about 5mm；
With being coated buffer solution, by sheep anti-mouse igg antibody, (Shenzhen City Fapon Biotech Co., Ltd produces, article No.
BA-PAB-MU0001) it is diluted to 1～5mg/mL, adjusts machine, be scribed ss C line, be control line,
C line is near absorption pad, away from absorption pad about 3mm.Two linear distances 5～8mm, uniformly.37 DEG C of drying, encapsulation
Standby.
B. collaurum, the preparation of gold labeled monoclonal antibody
(1) preparation of solution
1. the preparation of gold chloride: dissolve gold chloride by double distilled deionized water, is made into 1% solution, put 4 DEG C standby
With, the term of validity four months.1000mL1% chlorauric acid solution is filled a prescription: 10g gold chloride: double distilled deionized water is fixed
Hold to 1000mL.
2. the preparation of trisodium citrate: dissolve sodium citrate by double distilled deionized water, is made into 1% solution,
0.22 μm membrane filtration mistake, put 4 degree standby, the term of validity is held to 1000mL.
3. the preparation of 0.1M potassium carbonate: with double distilled deionized water prepare, 0.22 μm membrane filtration mistake, put 4 degree standby
With, the term of validity four months.1000mL0.1M solution of potassium carbonate formula: 13.8g potassium carbonate；Double boil off ion
Water is settled to 1000mL.
4. the preparation of 2%PEG-20000: prepare by double distilled deionized water, 0.22 μm membrane filtration mistake, put 4 DEG C
Standby, the term of validity four months.1000mL2%PEG-20000 solution formula: 20g PEG-20000；Double steamings
Deionized water is settled to 1000mL.
5. the preparation of mark washing preservation liquid: 2% bovine serum albumin(BSA) (BSA), 0.05% Sodium azide
(NaN3), 0.01M pH7.2PBS solution, 0.22 μ membrane filtration mistake, put 4 DEG C standby, the term of validity four months.
1000mL mark washing preservation formula of liquid: 20g BSA, 0.5g NaN3, 0.01M pH7.2PBS solution fixed
Hold to 1000mL.
(2) preparation of collaurum:
By double distilled deionized water, 1% gold chloride is diluted to 0.01%, puts electric furnace and boil, by every 100mL
0.01% gold chloride adds 2mL1% trisodium citrate, continues to boil, until liquid is that shiny red i.e. stops adding
Heat, supplies dehydration after being cooled to room temperature.The collaurum outward appearance prepared should be pure, bright, without precipitation and drift
Float, the term of validity one week.
(3) preparation of colloid gold label monoclonal antibody:
Adjust the pH value of collaurum to 8.2 with 0.1M potassium carbonate, add by 8～10 μ g antibody/mL collaurum real
Execute the anti-cardiac muscle troponin I monoclonal antibody prepared in example 1, magnetic stirring apparatus mixing 30min, stir
Mix lower addition BSA and stand 1 hour to final concentration of 1%.13000rpm, 4 DEG C of centrifugal 30min, abandon supernatant,
Precipitation mark washing preserves liquid and washes twice, and washs with the mark of 1/10th initial colloid gold volumes and preserves
Liquid will precipitate resuspended, put 4 DEG C standby, the term of validity one week.
C. the preparation of gold mark pad
(1) preparation of confining liquid:
2%BSA, 0.1%TritonX-100,0.05%NaN3, 0.01M pH7.2PBS solution, 0.22 μm
Membrane filtration mistake, put 4 degree standby, the term of validity four months.1000mL confining liquid is filled a prescription: 20g BSA, 0.5g NaN3、
1mL TritonX-100,0.01M pH7.2PBS solution are settled to 1000mL.
(2) preparation of gold mark pad:
Gold is marked pad and is soaked in confining liquid after 30min, in 37 DEG C of drying.Then the gold mark that will prepare
Antibody is layered on gold mark pad uniformly, and every milliliter of solution spreads 20 square centimeters, freeze-drying, and encapsulation puts 4 DEG C
Standby.
D. the preparation of test strips sample pad
(1) preparation of confining liquid:
2%BSA, 0.1%TrtionX-100,0.05%NaN3, 0.01M pH7.2PBS solution, 0.22 μm
Membrane filtration mistake, put 4 degree standby, the term of validity four months.1000mL confining liquid is filled a prescription: 20g BSA, 0.5g NaN3、
1mL TrtionX-100,0.01M pH7.2PBS solution are settled to 1000mL.
(2) preparation of sample pad:
Sample pad is soaked in confining liquid after 30min, in 37 DEG C of drying, encapsulation, put 4 DEG C standby.
E. the assembling of Test paper
Absorption pad (purchased from Millipore company), nitrocellulose filter, gold mark pad, sample pad are arranged on not
On the support slice of water suction, it is cut into the wide little bar of 3mm.Every ten little bars one wrap, and add drier, vacuum seal
Dress, obtains described Test paper.
The positive sample detected with Roche cardiac muscle troponin I diagnostic kit (Troponin I STAT) and the moon
Property sample as the detection sample of this kit, wherein 205 example cardiac muscle troponin Is detection positive sample, 800
Example detection negative sample, is shown in using the detection kit based on mouse source cTnI-C4 antibody as comparison, testing result
Table 2.Result shows, this kit detection positive sample 201 parts, relative sensitivity is 98.05%, with right
According to mouse source cTnI-C4 antibody test result consistent；800 parts of negative sample detect 788 parts, wherein 12 parts
Being false sun, relative specificity is 98.5%, and mouse source cTnI-C4 antibody detects 771 parts, and wherein 29 parts are
False sun, relative specificity is 96.38%, so restructuring cTnI-C4 antibody is applied to cardiac muscle troponin I
Diagnosis had both maintained the affinity of parent murine antibody, reduces false positive rate simultaneously, improves the special of diagnosis
Property, it is better than cardiac muscle troponin I diagnostic kit based on mouse source antibody.
Table 2 cardiac muscle troponin I based on mouse source antibody and recombinant antibodies detection kit testing result
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed,
But therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that, for this area
Those of ordinary skill for, without departing from the inventive concept of the premise, it is also possible to make some deformation and
Improving, these broadly fall into protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be with appended
Claim is as the criterion.
Claims (6)
1. the recombinant antibodies of an anti-human cardiac muscle troponin I, it is characterised in that include by anti-human myocardium myo
Light chain that the variable region of light chain of the mouse source antibody of calcium protein I is constituted with the constant region of light chain of human IgG1 and by anti-human
The heavy chain that the CH of the variable region of heavy chain of the mouse source antibody of cardiac muscle troponin I and human IgG1 is constituted；
Wherein, the mouse source antibody of described anti-human cardiac muscle troponin I is CCTCC C2013185 by preserving number
Hybridoma produce；
Described variable region of light chain contains the amino acid sequence as shown in SEQ ID NO.1；
Described variable region of heavy chain contains the amino acid sequence as shown in SEQ ID NO.2.
2. the construction method of the recombinant antibodies of an anti-human cardiac muscle troponin I, it is characterised in that include as
Lower step:
Build the expression containing the nucleotide sequence as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively to carry
Body, wherein, the expression vector containing the nucleotide sequence shown in SEQ ID NO.3 contains the light chain of human IgG1
The expressing gene of constant region, the expression vector containing the nucleotide sequence shown in SEQ ID NO.4 contains people
The expressing gene of the CH of IgG1；
Described expression vector is transfected in same host cell；
Reclaim from described host cell and obtain described anti-human cardiac muscle troponin I recombinant antibodies；
Wherein, be inserted into the expression vector of the nucleotide sequence as shown in SEQ ID NO.3 be reserved with XbaI and
PmlI double enzyme site；The expression vector being inserted into the nucleotide sequence as shown in SEQ ID NO.4 is reserved with
NheI and HindIII double enzyme site；
Described nucleotide sequence as shown in SEQ ID NO.3 is to be made by the steps acquisition:
From the hybridoma that preserving number is CCTCC C2013185, extract RNA, use Reverse Transcription box
Carry out after 70 DEG C of enzymes of RT-PCR, RT-PCR amplified production inactivate as template, with SEQ ID NO.5 and SEQ
Primer sequence shown in ID NO.6 carries out PCR, reclaims PCR primer, and PCR primer rTaq DNA gathers
Synthase is inserted in pMD-18T carrier after carrying out adding A reaction, is transformed in DH5 α competent cell expression；
Reclaim the product that DH5 α competent cell is expressed, use such as SEQ ID NO.7 and SEQ ID NO.8
Shown primer sequence carries out PCR, obtains the nucleotide sequence shown in described SEQ ID NO.3；
Described nucleotide sequence as shown in SEQ ID NO.4 is to be made by the steps acquisition:
From the hybridoma that preserving number is CCTCC C2013185, extract RNA, use Reverse Transcription box
Carry out after 70 DEG C of enzymes of RT-PCR, RT-PCR amplified production inactivate as template, with SEQ ID NO.9 and SEQ
Primer sequence shown in ID NO.10 carries out PCR, reclaims PCR primer, and PCR primer rTaq DNA gathers
Synthase is inserted in pMD-18T carrier after carrying out adding A reaction, is transformed in DH5 α competent cell expression；
Reclaim the product that DH5 α competent cell is expressed, use such as SEQ ID NO.11 and SEQ ID NO.12
Shown primer sequence carries out PCR, obtains the nucleotide sequence shown in described SEQ ID NO.4.
3. recombinant antibodies as claimed in claim 1 is in preparation detection cardiac muscle troponin I detection reagent
Application.
4. the Test paper of a cardiac muscle troponin I, it is characterised in that include as claimed in claim 1
Recombinant antibodies.
5. the Test paper of cardiac muscle troponin I as claimed in claim 4, it is characterised in that include propping up
Support thin slice, sample pad, gold mark pad, nitrocellulose filter, absorption pad, detection line and nature controlling line, described sample
Product pad, described gold mark pad, described nitrocellulose filter and described absorption pad from one end of described support slice to
The other end is successively set on described support slice, and described sample pad partly overlaps with described gold mark pad, described
Gold mark pad partly overlaps with described nitrocellulose filter, described nitrocellulose filter and described absorption pad part weight
Folded, described detection line and described nature controlling line are located on described nitrocellulose filter, and described detection line is located at and leans on
One end of nearly described gold mark pad, described nature controlling line is located at the one end near described absorption pad, on described gold mark pad
It is coated with the described attached colloid gold particle of recombinant antibodies bag and forms the recombinant antibodies of colloid gold label, described detection line
Rabbit for the anti-cardiac muscle troponin I of affinity purification resists more, and described nature controlling line is sheep anti-mouse igg antibody.
6. a detection kit, including the detection of the cardiac muscle troponin I as described in claim 4 or 5
Test paper.
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| CN105601744B (en) * | 2015-12-22 | 2020-08-04 | 广东唯实生物技术有限公司 | Recombinant antibody of anti-influenza A virus nucleoprotein, preparation method and application thereof |
| CN110878116B (en) * | 2018-09-05 | 2021-03-30 | 湖南永和阳光生物科技股份有限公司 | Stable recombinant cardiac troponin, and coding gene and application thereof |
| CN111018976B (en) * | 2018-10-10 | 2022-04-01 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-human cardiac troponin I |
| CN111018973B (en) * | 2018-10-10 | 2022-04-01 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-human cardiac troponin I |
| CN111018981B (en) * | 2018-10-10 | 2021-12-03 | 东莞市朋志生物科技有限公司 | Anti-human cardiac troponin I antibody and application thereof |
| CN111018982B (en) * | 2018-10-10 | 2021-12-03 | 东莞市朋志生物科技有限公司 | Anti-human cardiac troponin I antibody and application thereof |
| CN111018977B (en) * | 2018-10-10 | 2022-04-01 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-human cardiac troponin I |
| CN111018974B (en) * | 2018-10-10 | 2022-04-01 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-human cardiac troponin I |
| CN111018979B (en) * | 2018-10-10 | 2021-12-03 | 东莞市朋志生物科技有限公司 | Anti-human cardiac troponin I antibody and application thereof |
| CN111018980B (en) * | 2018-10-10 | 2021-12-03 | 东莞市朋志生物科技有限公司 | Anti-human cardiac troponin I antibody and application thereof |
| CN111018975B (en) * | 2018-10-10 | 2022-04-01 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-human cardiac troponin I |
| CN114206920B (en) * | 2019-08-09 | 2024-06-07 | 豪夫迈·罗氏有限公司 | Novel anti-troponin T antibodies |
| CN111308084B (en) * | 2019-12-30 | 2021-04-13 | 菲鹏生物股份有限公司 | Detection method and kit for hypersensitive cardiac troponin I |
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