CN110305874A - Meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its application - Google Patents
Meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its application Download PDFInfo
- Publication number
- CN110305874A CN110305874A CN201910533984.2A CN201910533984A CN110305874A CN 110305874 A CN110305874 A CN 110305874A CN 201910533984 A CN201910533984 A CN 201910533984A CN 110305874 A CN110305874 A CN 110305874A
- Authority
- CN
- China
- Prior art keywords
- meriones unguiculatus
- recombinant protein
- igg2
- immunoglobulin
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
- G01N33/686—Anti-idiotype
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/524—CH2 domain
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/526—CH3 domain
Abstract
The invention discloses meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its applications.Meriones unguiculatus Immunoglobulin IgG1 recombinant protein, including the histidine tag of meriones unguiculatus Immunoglobulin IgG1 heavy chain constant region CH2, CH3 and N-terminal, amino acid sequence is as shown in SEQ ID No.3, and nucleotide sequence is as shown in SEQ ID No.2.Meriones unguiculatus Immunoglobulin IgG2 recombinant protein, including the histidine tag of meriones unguiculatus Immunoglobulin IgG2 heavy chain constant region CH2, CH3 and N-terminal, amino acid sequence is as shown in SEQ ID No.4, and nucleotide sequence is as shown in SEQ ID No.1.Recombinant protein is used for Antibody preparation as immunizing antigen, or is used for immune detection.Recombinant protein provided by the present invention has the characteristics that high specificity, reproducible and with high purity compared with routine isolates and purifies IgG from gerbil jird serum.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of meriones unguiculatus Immunoglobulin IgG1 and IgG2 heavy chain are permanent
Determine the preparation and its application of area's CH2 and CH3 recombinant protein.
Background technique
Meriones unguiculatus (Meriones unguiculatus, Mongolian gerbil) size between rat and mouse it
Between, have the long period as experimental animal in medical domain, it is susceptible to a variety of cause of diseases, dynamic compared to the experiment of other rodents
Object, meriones unguiculatus are therefore close to can express pathological characteristics and clinical symptoms more like with the mankind after certain pathogen infections
Meriones unguiculatus has been increasingly being applied to the research fields such as multiple-microorganism (bacterium/virus) and parasitic infection over year.So
And still for the foundation of amynologic index of the meriones unguiculatus after pathogen infection especially specific antibody Serologic detection system
There are many places to be improved.In other words, specific antibody/immune globulin of generation is induced after exogenous pathogen infection meriones unguiculatus
The detection of white (Immunoglobulin, Ig) such as IgG or IgM still lacks corresponding specific secondary antibody, that is, antiantibody, thus to length
Research in terms of pawl Gerbil immunizations response especially humoral immune response, which receives, significantly to be limited.
To meriones unguiculatus antibody generate detection initially (2002) using indirect agglutinating reaction methods and utilize anti-mouse
The enzyme-linked immuno-sorbent assay (ELISA) of IgG polyclonal antibody, thus the accuracy of experimental result and special may be influenced
Property.On the other hand, different study group domestic in recent years is using in Protein G affinitive layer purification meriones unguiculatus serum
Then IgG is used to prepare rabbit anti-serum/polyclonal antibody as immunogene.IgM content is compared with it in meriones unguiculatus serum
Its animal is high, and the content of IgG is not high.Since Protein G can also adsorb other antibody in meriones unguiculatus serum point
Son such as IgM, thus they purify and are likely to be mingled with containing other antibody molecules such as IgM etc. in the meriones unguiculatus IgG of acquisition.Cause
This, the rabbit-anti meriones unguiculatus IgG polyclonal antibody that they prepare is also likely to intersect the identification other antibody molecules of meriones unguiculatus such as
IgM etc., so that it be caused to be applied to the experimental result deviation of such as ELISA in next step.
Ukaji etc. determines coding 291 amino acid of meriones unguiculatus IgG1 heavy chain constant region and IgG2 heavy chain constant region 251
Partial gene sequence [Ukaji T, Exp Anim, 2012,61 (2): 99-107 of a amino acid；Ukaji T, Anim Sci
J, 2011, 82(5):713-716.].Muroid IgG heavy chain constant region albumen/amino acid sequence comparative analysis shows meriones unguiculatus
Identity between IgG1 heavy chain constant region and IgG2 heavy chain constant region is 72.4%, the former and 1 heavy chain constant region of mouse IgG
Identity is up to 78.0%, and the identity of the IgG heavy chain constant region of the latter and hamster is up to 84.6%.Although meriones unguiculatus
IgG1 and IgG2 heavy chain constant region respectively with mouse IgG 1, IgG2a, IgG2b and IgG3 heavy chain constant region have 67.0%~
78.0% and 78.1%~81.8% identity, but commercially available rabbit or goat anti-mouse igg polyclonal antibody can not intersect identification length
Pawl gerbil jird IgG [Zhang Feng, Chinese comparative medicine magazine, 2017,27 (1): 37-42].
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of meriones unguiculatus IgG1 and IgG2 heavy chain constant region CH2 and
The preparation and its application of CH3 recombinant protein.
A kind of gene of meriones unguiculatus Immunoglobulin IgG1 recombinant protein, including meriones unguiculatus Immunoglobulin IgG1 weight
The histidine tag of chain constant region CH2, CH3 and N-terminal, nucleotide sequence is as shown in SEQ ID No.1.
A kind of meriones unguiculatus Immunoglobulin IgG1 recombinant protein, including meriones unguiculatus Immunoglobulin IgG1 light chain constant
The histidine tag of area CH2, CH3 and N-terminal, amino acid sequence is as shown in SEQ ID No.3.
A kind of gene of meriones unguiculatus Immunoglobulin IgG2 recombinant protein, including meriones unguiculatus Immunoglobulin IgG2 weight
The histidine tag of chain constant region CH2, CH3 and N-terminal, nucleotide sequence is as shown in SEQ ID No.2.
A kind of meriones unguiculatus Immunoglobulin IgG2 recombinant protein, including meriones unguiculatus Immunoglobulin IgG2 light chain constant
The histidine tag of area CH2, CH3 and N-terminal, amino acid sequence is as shown in SEQ ID No.4.
The gene of one group long pawl Gerbil immunizations globulin IgG1 and IgG2 recombinant protein, comprising: meriones unguiculatus immune globulin
The histidine tag of white IgG1 heavy chain constant region CH2, CH3 and N-terminal, nucleotide sequence is as shown in SEQ ID No.1；Long pawl is husky
The histidine tag of rat immune globulin IgG2 heavy chain constant region CH2, CH3 and N-terminal, nucleotide sequence such as SEQ ID No.2
It is shown.
One group long pawl Gerbil immunizations globulin IgG1 and IgG2 recombinant protein, comprising: meriones unguiculatus Immunoglobulin IgG1
The histidine tag of heavy chain constant region CH2, CH3 and N-terminal, amino acid sequence is as shown in SEQ ID No.3；Meriones unguiculatus is immune
The histidine tag of globulin IgG2 heavy chain constant region CH2, CH3 and N-terminal, amino acid sequence is as shown in SEQ ID No.4.
The application of the recombinant protein is used for Antibody preparation as immunizing antigen, or is used for immune detection.
The application of the gene of the recombinant protein is cloned into expression plasmid pET-28a (+).
Beneficial effects of the present invention:
Using meriones unguiculatus IgG1 and IgG2 heavy chain constant region CH2 and CH3 recombinant protein as immunogene, spy has been prepared
Anisotropic polyclonal antibody can be used as secondary antibody for purposes such as MBP enzyme linked immuno-adsorbent assays.For meriones unguiculatus specific antibody serum
It learns detection and provides effective tool, facilitate standardization and popularization and use of the meriones unguiculatus as experimental animal.
Detailed description of the invention
Fig. 1 is recombinant expression plasmid coding schematic diagram；
Fig. 2 is the expression and purification product electrophoresis and western blot figure of gerbil jird IgG1CH23 recombinant protein；
Fig. 3 is the expression and purification product electrophoretogram and western blot figure of gerbil jird IgG2CH23 recombinant protein；
Fig. 4 is rabbit-anti gerbil jird IgG1CH23 and IgG2CH23 polyclonal antibody electrophoretogram；
Fig. 5 is rabbit-anti gerbil jird IgG1CH23 and IgG2CH23 polyclonal antibody is used for Western Blot immunodetection technology (WB)；
Fig. 6 is rabbit-anti gerbil jird IgG1CH23 and IgG2CH23 polyclonal antibody is used for MBP enzyme linked immuno-adsorbent assay (ELISA).Figure
In, 1+2: primary antibody is the anti-CVA16 serum of gerbil jird, secondary antibody is HPP label rabbit-anti gerbil jird IgG1CH23 and IgG2CH23 Anti-TNF-α
Body；1: primary antibody is the anti-CVA16 serum of gerbil jird, secondary antibody is HPP label rabbit-anti gerbil jird IgG1CH23 polyclonal antibody；2: primary antibody is sand
The anti-CVA16 serum of mouse, secondary antibody are that HPP marks rabbit-anti gerbil jird IgG2CH23 polyclonal antibody；C: primary antibody is gerbil jird non-immune serum
Control, secondary antibody are that HPP marks rabbit-anti gerbil jird IgG1CH23 and IgG2CH23 polyclonal antibody.
Specific embodiment
The present invention is further elaborated with attached drawing with reference to embodiments.
Recombinant protein, including meriones unguiculatus Immunoglobulin IgG1 heavy chain constant region CH2, CH3 or IgG2 light chain constant
Area CH2, CH3.Above-mentioned recombinant protein is according to reported meriones unguiculatus IgG1 heavy chain constant region (GenBank sequence number
AB663132) and IgG2 heavy chain constant region (GenBank sequence number AB597231), CH2 and CH3 section therein is chosen.In order to
Be conducive to recombinant protein in E. coli, by its corresponding gene order carry out codon optimization and N-terminal with
Histidine (His) tag fusion is cloned into expression plasmid pET-28a (+) and obtains recombinant plasmid pET-28a (+)-gerbil jird
IgG1CH23 and pET-28a (+)-gerbil jird IgG2-CH23, converts e. coli bl21 (DE3) competent cell, and screening is turned
Beggar recombinantly expresses bacterium.Recombinant protein is inoculated in rabbit through inducing expression and after purification, as immunogene, after obtaining antiserum
Specific polyclonal antibody is obtained through affinity antibody column chromatography.
For the convenience of recombinant protein purification, affinity purification label can be used.The recombinant protein includes histidine
(His) sequence label.
The recombinant protein, the amino acid sequence of meriones unguiculatus Immunoglobulin IgG1 heavy chain constant region CH2 and CH3 are such as
Shown in SEQ ID No.3, nucleotide sequence is as shown in SEQ ID No.1.
The amino acid sequence such as SEQ ID No.4 institute of meriones unguiculatus Immunoglobulin IgG2 heavy chain constant region CH2 and CH3
Show, nucleotide sequence is as shown in SEQ ID No.2.
The structure of embodiment 1 recombinant plasmid pET-28a (+)-gerbil jird IgG1CH23 and pET-28a (+)-gerbil jird IgG2-CH23
It builds and converts
According to the protein sequence of meriones unguiculatus Immunoglobulin IgG1 and IgG2 heavy chain constant region, their own constant domain is selected
CH2 and CH3, by artificial-synthetic DNA's segment after codon optimization, both ends are directed respectively into corresponding digestion with restriction enzyme position
Point NcoI and XhoI is simultaneously cloned into colibacillus expression plasmid pET-28a (+), makes the N-terminal of destination protein with His label with benefit
In purifying, it is identified correct to obtain corresponding recombinant plasmid.Recombinant expression plasmid coding schematic diagram is as shown in Figure 1.
Recombinant plasmid pET-28a (+)-IgG1CH23 expresses the constant domain CH2 and CH3 of gerbil jird IgG1, encodes 218 amino
Acid, 24.8 kDa of theoretical molecular weight；Recombinant plasmid pET-28a (+)-IgG2CH23 express gerbil jird IgG2 constant domain CH2 and
CH3 encodes 187 amino acid, 21.2 kDa of theoretical molecular weight.
By recombinant plasmid transformed e. coli bl21 (DE3) competent cell, screening obtains transformant and recombinantly expresses bacterium.
The expression of 2 meriones unguiculatus IgG1-CH23 and IgG2-CH23 recombinant protein of embodiment and purifying
Recombinant plasmid pET-28a (+)-IgG1CH23 and pET-28a (+)-IgG2-CH23 converts e. coli bl21 respectively
(DE3) competent cell is laid on 37 °C of overnight incubations of LB plate, chooses single colonie respectively and be inoculated in LB liquid medium, culture
To absorbance A600Up to after 0.8, IPTG(1 mmol/L is added) 25 °C of overnight inductions.
Next day is collected by centrifugation coli somatic and is resuspended in the broken bacterium of ultrasound after buffer, and supernatant precipitating is collected by centrifugation, uses
The expression-form of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis recombinant protein.Broken bacterium is precipitated molten
In the liquid of urea containing 8mol/L, supernatant is collected after centrifugation, His GravitrapTM column is crossed under Denaturing, utilizes imidazole concentration
Gradient elution simultaneously collects destination protein, then in dialyzate (10 mmol/L Tris, 20 mmol/L NaCl [pH9.0])
4 °C of dialysis renaturations simultaneously gradually remove urea.
The expression and purification product electrophoresis of gerbil jird IgG1CH23 recombinant protein and western blot figure such as Fig. 2 institute
Show, the expression and purification product electrophoresis and western blot figure of gerbil jird IgG2CH23 recombinant protein are as shown in Figure 3.
After recombinant plasmid converts Escherichia coli respectively, through IPTG induction can high efficiency expressing destination protein, all with inclusion body
Form exists and molecular size range is consistent with expection.The two N-terminal introduces His label all in favor of purifying, thus WB with it is anti-
His antibody is reacted in specific positive.
The preparation of 3 rabbit-anti meriones unguiculatus IgG1-CH23 and IgG2-CH23 polyclonal antibody of embodiment
Recombinant protein meriones unguiculatus IgG1 CH23 and the IgG2 CH23 of above-mentioned purifying is inoculated in new west as immunogene
Blue large ear rabbit is sub, subcutaneous multi-point injection, takes a blood sample after initial immunity four weeks, and it is simultaneously affine with Hitrap Protein A to separate serum
Antibody column chromatography obtains polyclonal antibody.
Rabbit-anti gerbil jird IgG1CH23 or IgG2CH23 polyclonal antibody electrophoretogram is as shown in Figure 4.
4 rabbit-anti meriones unguiculatus IgG1 CH23 and IgG2 CH23 polyclonal antibody of embodiment is used for Western Blot immunodetection
Technology (WB)
Above-mentioned rabbit-anti meriones unguiculatus IgG1 CH23 and IgG2 CH23 polyclonal antibody is used into horseradish peroxidase (HRP) respectively
After label, it can specifically identify that the long pawl of the recombinant protein of Bacillus coli expression is husky for Western Blot immunodetection technology (WB)
Mouse IgG1 CH23 and IgG2 CH23(Fig. 5).
5 rabbit-anti meriones unguiculatus IgG1 CH23 and IgG2 CH23 polyclonal antibody of embodiment is examined for Enzyme-linked Immunosorbent Assay
It surveys (ELISA)
By YY157 plants of coxsackie virus A 16-type of inactivation as capture antigen coat plates, the anti-CVA16 serum of meriones unguiculatus or
Non-immune serum is compareed as primary antibody, by rabbit-anti meriones unguiculatus IgG1 CH23 and IgG2 the CH23 polyclonal antibody of HRP label
It is used for ELISA as secondary antibody/anti-igg antibody, induces production after coxsackie virus A 16-type (CAV16) infection meriones unguiculatus can be detected
Raw anti-CAV16 specific IgG antibodies (Fig. 6).
Above-described embodiment the result shows that, using molecular cloning means express respectively meriones unguiculatus IgG1CH2 and CH3 recombination egg
White and IgG2 heavy-chain constant domains CH2 and CH3 recombinant protein, and successfully prepared specific rabbit polyclonal as immunogene and resisted
Body provides effective tool for meriones unguiculatus specific antibody Serologic detection, facilitates meriones unguiculatus as experimental animal
Standardization and popularization and use.
Sequence table
<110>Zhejiang Prov. Tumor Hospital
<120>meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its application
<141> 2019-06-19
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 665
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ccatgggtca tcaccaccac catcacggcc tggttccgcg tggcagcgtg ccggaagttt 60
cttccgtgtt catctttcca ccgaaaccga aagatgttct gactatctcc ctgactccga 120
agatcacctg cgtggtggtt gacatcagcc aggacgatcc agaggtggag atcagctggt 180
tcgttgacga tgtggaagta cacactgccc agacccagcc acgtgaagaa cagtttaact 240
ctactttccg tgtggtatcc accctgccga tcctgcacca ggactggctg aatggtaaag 300
agttcaaatg caaagtgaac agcgaagcat ttccgagccc aatcgagaag accgtgagca 360
agatgaaagg tcagccgcag gcaccgcagg tttacatgat gccaccaccg aaagagcaga 420
tgactaagaa cgaggtaact gtttcctgct acgttggcgg tttctatccg ccagatgttt 480
ccgtagagtg gcagcgcaat ggcgagccgg aaccgaactt caaatctacc cagccgaccc 540
tggacactga cgagtcctac ttcctgtact ctaaactgga tgtaaagcgt gcaaactggg 600
agaaaggtga cgtgttcact tgctccgtgc tgcacgaggc actgcacaat catcactaac 660
tcgag 665
<210> 2
<211> 572
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccatgggcca tcaccaccac catcacggtc tggttccgcg tggcagcgct ccagacctgt 60
ctggcggtcc gtccgtgttt atcttcccac caaagccgaa agatgtgctg atgatgaccc 120
tgactccgaa gatcacctgc gtggtagtag acgtttccga ggacgaaccg gatgttcagt 180
tcaactggtt cgttgacaat gtggaagtgc ataacgcgca gacccaaccg cgtgagggtc 240
agtacaactc taccttccgt gtagtgtctg cgctgccgat taagcaccaa gactggatgt 300
ctggcaaaga gttcaaatgc aaagttaaca acaaagcact gccgtctccg atcgagaaga 360
ccatttccaa gccaaagggt ccggttcgtg agccgcaggt ttacgccttt ccaccaccga 420
aggaacagat gaaccgtaac cagctgtctc tgacctgcat gattaccaac ttctatccgg 480
aagcgatcga cgtggaatgg gaacgcaacg gccaggaaga gcgtaactac aagaacacca 540
aaccagtgct ggattccgat ggctaactcg ag 572
<210> 3
<211> 218
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Met Gly His His His His His His Gly Leu Val Pro Arg Gly Ser Val
1 5 10 15
Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val
20 25 30
Leu Thr Ile Ser Leu Thr Pro Lys Ile Thr Cys Val Val Val Asp Ile
35 40 45
Ser Gln Asp Asp Pro Glu Val Glu Ile Ser Trp Phe Val Asp Asp Val
50 55 60
Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Phe Arg Val Val Ser Thr Leu Pro Ile Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Phe Lys Cys Lys Val Asn Ser Glu Ala Phe Pro Ser
100 105 110
Pro Ile Glu Lys Thr Val Ser Lys Met Lys Gly Gln Pro Gln Ala Pro
115 120 125
Gln Val Tyr Met Met Pro Pro Pro Lys Glu Gln Met Thr Lys Asn Glu
130 135 140
Val Thr Val Ser Cys Tyr Val Gly Gly Phe Tyr Pro Pro Asp Val Ser
145 150 155 160
Val Glu Trp Gln Arg Asn Gly Glu Pro Glu Pro Asn Phe Lys Ser Thr
165 170 175
Gln Pro Thr Leu Asp Thr Asp Glu Ser Tyr Phe Leu Tyr Ser Lys Leu
180 185 190
Asp Val Lys Arg Ala Asn Trp Glu Lys Gly Asp Val Phe Thr Cys Ser
195 200 205
Val Leu His Glu Ala Leu His Asn His His
210 215
<210> 4
<211> 187
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Met Gly His His His His His His Gly Leu Val Pro Arg Gly Ser Ala
1 5 10 15
Pro Asp Leu Ser Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro
20 25 30
Lys Asp Val Leu Met Met Thr Leu Thr Pro Lys Ile Thr Cys Val Val
35 40 45
Val Asp Val Ser Glu Asp Glu Pro Asp Val Gln Phe Asn Trp Phe Val
50 55 60
Asp Asn Val Glu Val His Asn Ala Gln Thr Gln Pro Arg Glu Gly Gln
65 70 75 80
Tyr Asn Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Lys His Gln
85 90 95
Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Ala
100 105 110
Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro Lys Gly Pro Val
115 120 125
Arg Glu Pro Gln Val Tyr Ala Phe Pro Pro Pro Lys Glu Gln Met Asn
130 135 140
Arg Asn Gln Leu Ser Leu Thr Cys Met Ile Thr Asn Phe Tyr Pro Glu
145 150 155 160
Ala Ile Asp Val Glu Trp Glu Arg Asn Gly Gln Glu Glu Arg Asn Tyr
165 170 175
Lys Asn Thr Lys Pro Val Leu Asp Ser Asp Gly
180 185
Claims (8)
1. a kind of gene of meriones unguiculatus Immunoglobulin IgG1 recombinant protein, which is characterized in that ball is immunized including meriones unguiculatus
The histidine tag of protein I gG1 heavy chain constant region CH2, CH3 and N-terminal, nucleotide sequence is as shown in SEQ ID No.1.
2. a kind of meriones unguiculatus Immunoglobulin IgG1 recombinant protein, which is characterized in that including meriones unguiculatus immunoglobulin
The histidine tag of IgG1 heavy chain constant region CH2, CH3 and N-terminal, amino acid sequence is as shown in SEQ ID No.3.
3. a kind of gene of meriones unguiculatus Immunoglobulin IgG2 recombinant protein, which is characterized in that ball is immunized including meriones unguiculatus
The histidine tag of protein I gG2 heavy chain constant region CH2, CH3 and N-terminal, nucleotide sequence is as shown in SEQ ID No.2.
4. a kind of meriones unguiculatus Immunoglobulin IgG2 recombinant protein, which is characterized in that including meriones unguiculatus immunoglobulin
The histidine tag of IgG2 heavy chain constant region CH2, CH3 and N-terminal, amino acid sequence is as shown in SEQ ID No.4.
5. the gene of one group long pawl Gerbil immunizations globulin IgG1 and IgG2 recombinant protein characterized by comprising meriones unguiculatus
The histidine tag of Immunoglobulin IgG1 heavy chain constant region CH2, CH3 and N-terminal, nucleotide sequence such as SEQ ID No.1 institute
Show；The histidine tag of meriones unguiculatus Immunoglobulin IgG2 heavy chain constant region CH2, CH3 and N-terminal, nucleotide sequence such as SEQ
Shown in ID No.2.
6. one group long pawl Gerbil immunizations globulin IgG1 and IgG2 recombinant protein characterized by comprising ball is immunized in meriones unguiculatus
The histidine tag of protein I gG1 heavy chain constant region CH2, CH3 and N-terminal, amino acid sequence is as shown in SEQ ID No.3；Long pawl
The histidine tag of Gerbil immunizations globulin IgG2 heavy chain constant region CH2, CH3 and N-terminal, amino acid sequence such as SEQ ID
Shown in No.4.
7. such as the described in any item recombinant protein applications of claim 2,4,6, which is characterized in that be used for antibody as immunizing antigen
Preparation, or it is used for immune detection.
8. such as the gene application of the described in any item recombinant proteins of claim 1,3,5, which is characterized in that be cloned into expression plasmid
pET-28a(+)。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910533984.2A CN110305874A (en) | 2019-06-19 | 2019-06-19 | Meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910533984.2A CN110305874A (en) | 2019-06-19 | 2019-06-19 | Meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110305874A true CN110305874A (en) | 2019-10-08 |
Family
ID=68076470
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910533984.2A Pending CN110305874A (en) | 2019-06-19 | 2019-06-19 | Meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110305874A (en) |
Citations (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1486192A (en) * | 2001-01-17 | 2004-03-31 | ��³�Ȱ�ҩƷ��˾ | Binding domain-immunoglobulin fusion proteins |
| US6891024B2 (en) * | 2001-05-24 | 2005-05-10 | The Curators Of The University Of Missouri | Monoclonal antibodies to Sarcocystis neurona and uses therefor |
| US20060148750A1 (en) * | 1999-12-22 | 2006-07-06 | Shiver John W | Polynucleotide vaccines expressing codon optimized HIV-1 Pol and modified HIV-1 Pol |
| CN101258164A (en) * | 2005-08-16 | 2008-09-03 | 韩美药品工业株式会社 | A method for the mass production of immunoglobulin fc region deleted initial methionine residues |
| AU2005201915B2 (en) * | 1999-05-28 | 2009-01-22 | Genentech, Inc. | DR4 antibodies and uses thereof |
| CN101575379A (en) * | 2008-05-09 | 2009-11-11 | 上海中信国健药业有限公司 | Soluble VEGFR difunctional fusion receptors, preparation method and use thereof |
| CN102036677A (en) * | 2008-03-20 | 2011-04-27 | 迈阿密大学 | Heat shock protein GP96 vaccination and methods of using same |
| CN102175867A (en) * | 2010-12-23 | 2011-09-07 | 北京民海生物科技有限公司 | Enzyme-linked immunoassay kit for detecting rabies virus antibody |
| CN103901202A (en) * | 2014-04-04 | 2014-07-02 | 中国人民解放军成都军区疾病预防控制中心 | Universal competitive ELISA detection method based on common epitope influenza A virus antibody |
| US20140245468A1 (en) * | 2013-02-20 | 2014-08-28 | Regeneron Pharmaceuticals, Inc. | Non-human animals with modified immunoglobulin heavy chain sequences |
| CN104076146A (en) * | 2014-06-19 | 2014-10-01 | 华北制药集团新药研究开发有限责任公司 | ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting human anti-rabies virus antibody |
| CN104262488A (en) * | 2014-09-24 | 2015-01-07 | 普莱柯生物工程股份有限公司 | Preparation and application of fusion protein and vaccine composition thereof |
| CN105137073A (en) * | 2015-08-06 | 2015-12-09 | 中国兽医药品监察所 | Bovine Brucella colloidal gold antibody detection test paper strip |
| CN105759038A (en) * | 2016-04-15 | 2016-07-13 | 肖乐义 | Immunoassay method and kit for assaying mycobacterium tuberculosis from biological samples |
| CN107011445A (en) * | 2007-06-01 | 2017-08-04 | 马里兰大学巴尔的摩分校 | Immunoglobulin constant region fc receptor binding agents |
| US20170226162A1 (en) * | 2016-02-04 | 2017-08-10 | Trianni, Inc | Enhanced production of immunoglobulins |
| CN107428830A (en) * | 2015-01-05 | 2017-12-01 | 依奈特制药公司 | Monomer FC domains |
| CN108205063A (en) * | 2016-12-20 | 2018-06-26 | 浙江特瑞思药业股份有限公司 | The detection method of a kind of anti-CD-20 monoclonal antibody and antigen-binding activity (filter membrane plate ELISA) |
| CN108226513A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | It is a kind of to detect the enzyme linked immunological kit that pseudo- cow's milk is mixed in buffalo's milk and sheep breast |
| CN109180817A (en) * | 2018-09-19 | 2019-01-11 | 程晓东 | A kind of novel anti-human igg 1-Fc antibody reagent and preparation method thereof |
-
2019
- 2019-06-19 CN CN201910533984.2A patent/CN110305874A/en active Pending
Patent Citations (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2005201915B2 (en) * | 1999-05-28 | 2009-01-22 | Genentech, Inc. | DR4 antibodies and uses thereof |
| US20060148750A1 (en) * | 1999-12-22 | 2006-07-06 | Shiver John W | Polynucleotide vaccines expressing codon optimized HIV-1 Pol and modified HIV-1 Pol |
| CN1486192A (en) * | 2001-01-17 | 2004-03-31 | ��³�Ȱ�ҩƷ��˾ | Binding domain-immunoglobulin fusion proteins |
| US6891024B2 (en) * | 2001-05-24 | 2005-05-10 | The Curators Of The University Of Missouri | Monoclonal antibodies to Sarcocystis neurona and uses therefor |
| CN101258164A (en) * | 2005-08-16 | 2008-09-03 | 韩美药品工业株式会社 | A method for the mass production of immunoglobulin fc region deleted initial methionine residues |
| CN107011445A (en) * | 2007-06-01 | 2017-08-04 | 马里兰大学巴尔的摩分校 | Immunoglobulin constant region fc receptor binding agents |
| CN102036677A (en) * | 2008-03-20 | 2011-04-27 | 迈阿密大学 | Heat shock protein GP96 vaccination and methods of using same |
| CN101575379A (en) * | 2008-05-09 | 2009-11-11 | 上海中信国健药业有限公司 | Soluble VEGFR difunctional fusion receptors, preparation method and use thereof |
| CN102175867A (en) * | 2010-12-23 | 2011-09-07 | 北京民海生物科技有限公司 | Enzyme-linked immunoassay kit for detecting rabies virus antibody |
| US20140245468A1 (en) * | 2013-02-20 | 2014-08-28 | Regeneron Pharmaceuticals, Inc. | Non-human animals with modified immunoglobulin heavy chain sequences |
| CN103901202A (en) * | 2014-04-04 | 2014-07-02 | 中国人民解放军成都军区疾病预防控制中心 | Universal competitive ELISA detection method based on common epitope influenza A virus antibody |
| CN104076146A (en) * | 2014-06-19 | 2014-10-01 | 华北制药集团新药研究开发有限责任公司 | ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting human anti-rabies virus antibody |
| CN104262488A (en) * | 2014-09-24 | 2015-01-07 | 普莱柯生物工程股份有限公司 | Preparation and application of fusion protein and vaccine composition thereof |
| CN107428830A (en) * | 2015-01-05 | 2017-12-01 | 依奈特制药公司 | Monomer FC domains |
| CN105137073A (en) * | 2015-08-06 | 2015-12-09 | 中国兽医药品监察所 | Bovine Brucella colloidal gold antibody detection test paper strip |
| US20170226162A1 (en) * | 2016-02-04 | 2017-08-10 | Trianni, Inc | Enhanced production of immunoglobulins |
| CN105759038A (en) * | 2016-04-15 | 2016-07-13 | 肖乐义 | Immunoassay method and kit for assaying mycobacterium tuberculosis from biological samples |
| CN108226513A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | It is a kind of to detect the enzyme linked immunological kit that pseudo- cow's milk is mixed in buffalo's milk and sheep breast |
| CN108205063A (en) * | 2016-12-20 | 2018-06-26 | 浙江特瑞思药业股份有限公司 | The detection method of a kind of anti-CD-20 monoclonal antibody and antigen-binding activity (filter membrane plate ELISA) |
| CN109180817A (en) * | 2018-09-19 | 2019-01-11 | 程晓东 | A kind of novel anti-human igg 1-Fc antibody reagent and preparation method thereof |
Non-Patent Citations (9)
| Title |
|---|
| ALEXEY TEPLYAKOV等: "IgG2 Fc Structure and the Dynamic Features of the IgG CH2-CH3 Interface", 《MOL IMMUNOL》 * |
| TAKAO UKAJI等: "Partial sequence of Mongolian gerbil (Meriones unguiculatus) immunoglobulin gamma heavy chain constant region", 《ANIMAL SCIENCE JOURNAL》 * |
| TAKAO UKAJI等: "Sequence Determination of the Heavy-Chain Constant Region in Four Immunoglobulin Classes of Mongolian Gerbils (Meriones Unguiculatus)", 《EXPERIMENTAL ANIMALS》 * |
| TIANLEI YING等: "Interactions of IgG1 CH2 and CH3 Domains With FcRn", 《FRONT IMMUNOL》 * |
| 刘亮伟等主编: "《基因工程原理与实验指导》", 30 September 2010, 中国轻工业出版社 * |
| 姜法铭等: "猪IgG H链基因CH2-CH3的克隆和分析", 《上海农业学报》 * |
| 李萍等: "HRP-SPA用于丙型肝炎检测 ", 《江苏大学学报(医学版)》 * |
| 陆曙梅: "《微生物学与免疫学基础》", 31 July 2007, 河南科学技术出版社 * |
| 顾青等主编: "《分子生物学实验指导》", 28 February 2014, 浙江工商大学出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102103857B1 (en) | Nc fusion protein comprising n-terminus domain fragments and c-terminus domain fragments of mers-cov nucleocapsid protein and kit for diagnosing infection of mers-cov using the same | |
| Ogun et al. | The oligomerization domain of C4-binding protein (C4bp) acts as an adjuvant, and the fusion protein comprised of the 19-kilodalton merozoite surface protein 1 fused with the murine C4bp domain protects mice against malaria | |
| US9644020B2 (en) | Human Tim-3 fusion protein capable of blocking Tim-3 signaling pathway | |
| CN112094326B (en) | New coronavirus antigen and application thereof | |
| KR101528169B1 (en) | Method and kit for detection of anti-avibacterium paragallinarum antibody | |
| CN113018427A (en) | Multivalent fusion protein vaccine based on neutralizing epitope of new coronavirus | |
| CN106279378A (en) | Varicella zoster virus gE antigen and the purposes in detection varicella zoster virus antibody thereof | |
| CN115093477A (en) | Monoclonal antibody for resisting N terminal region of novel coronavirus nucleoprotein and application thereof | |
| CN107033226B (en) | Peste des petits ruminants virus F protein epitope peptide, determination method, preparation method and application thereof | |
| WO2018199538A1 (en) | Fusion protein, in which hagfish-derived vlrb protein from which hydrophobic tail domain has been removed and c4bp oligomerization domain are bound, and use thereof | |
| KR100905066B1 (en) | The recombinant proteins for the diagnosis of diseases infected from Mycoplasma pneumoniae and the diagnostic kits comprising the same | |
| CN102702324A (en) | Application of human procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof | |
| CN111705066A (en) | Genetically modified TIGIT protein, monoclonal antibody and application thereof | |
| CN103725697A (en) | Chemically synthesized staphylococcus aureus surface protein FnBPA gene fragment and expression and application thereof | |
| CN110305874A (en) | Meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its application | |
| CN105602915A (en) | Multi-valence EZH2 tumor-associated antigen peptide and preparation thereof | |
| CN103880953A (en) | Porcine P21 protein antibody as well as preparation method and application thereof | |
| CN109400684A (en) | A kind of PEDV S-RBD Linear B Cell Epitopes and two plants of specific recognition monoclonal antibodies and application | |
| CN103848888B (en) | A kind of people's C1Q/TNF α GAP-associated protein GAP 2（hCTRP2）Antigenic Peptide and its antibody | |
| CN114163504A (en) | SARS-CoV-2S protein RBD zone neutralizing epitope peptide and its application | |
| CN102643332A (en) | Application of B cell epitope peptide of human PCT (Procalcitonin) and monoclonal antibody of B cell epitope peptide | |
| KR102259974B1 (en) | Method for producing target antigen-specific antibody using recombinant antigen | |
| CN103524601B (en) | Epitope peptide in human oocyte zona pellucida protein 4 and application thereof | |
| CN108084256A (en) | Procalcitonin mutant and its preparation method and application | |
| CN111925447B (en) | Human and mouse egg zona pellucida fusion protein and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191008 |
|
| RJ01 | Rejection of invention patent application after publication |