CN102174522B

CN102174522B - Preparation method of protein 4-1BBL

Info

Publication number: CN102174522B
Application number: CN 201110045245
Authority: CN
Inventors: 黄茵
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2011-02-24
Filing date: 2011-02-24
Publication date: 2013-11-06
Anticipated expiration: 2031-02-24
Also published as: CN102174522A

Abstract

The invention provides a preparation method of a protein 4-1BBL. In the preparation method, a recombinant protein 4-1BBL containing 206 amino acid residues (Arg 104-Glu 309) is prepared from an extracellular-segment protein of mouse 4-1BBL (TNFSF9 (Tumor Necrosis Factor Receptor Superfamily 9), NP_033430.1) by using a gene engineering method. By adopting the preparation method provided by the invention, a large quantity of proteins 4-1BBL can be obtained, so that the yield of the protein is greatly improved, and the obtained extracellular-segment protein 4-1BBL can be applied to preparation of kits for the quantitative pre-clinical researches of the protein 4-1BBL or for medicinal purposes and has a wide application prospect.

Description

A kind of preparation method of 4-1BBL albumen

(1) technical field

The present invention relates to a kind of preparation method of 4-1BBL albumen.

(2) background technology

4-1BB/4-1BBL is that another outside CD28/B7 is to important costimulatory signal molecule.4-1BB (CD137) belongs to Tumor Necrosis Factor Receptors (TNFR) superfamily, mainly be distributed in the surfaces such as T cell, NK cell and DC cell of activation, its part 4-1BBL (CD137L) is main to express on the T cell and some tumour cells of full-time APC cell (as mononuclear macrophage, DC cell, B cell), activation.4-1BBL is II type transmembrane glycoprotein, and molecular weight is 34KD, and people and mouse have 36% homology.

The 4-1BB/4-1BBL signal plays an important role to strengthening and keeping immunne response.Compare with the CD28/B7 signal transduction pathway, 4-1BB/4-1BBL is CD8 mainly in the later stage of immunne response ⁺The amplification of T cell clone, survival and memory are killed and wounded signal are provided, and it is more obvious that it stimulates acting on of T cell proliferation and secrete cytokines to show when lacking powerful CD28/B7 signal.The activation of 4-1BB is to CD4 ⁺And CD8 ⁺The T cell all has effect, but preferentially stimulates CD8 ⁺The activation of T cell.Except passing through 4-1BB receptor for stimulating T extracellular, 4-1BBL also stimulates monocyte by its reverse signal conduction path, causes M-CSF to express and discharges cytokine profiles, the prolongation monocyte survival phase, promotes cell proliferation and mitotic division.

In recent years, the 4-1BB/4-1BBL signal transduction pathway has become a novel targets of immunotherapy of tumors design.In body, experiment shows, the necrocytosis (AICD) that the 4-1BB monoclonal antibody can prevent activation to induce, and the propagation of promotion lotus knurl host medium size lymphocyte, and selectivity promotes the secretion of I cytokines, the antitumor action of reinforcing effect cell.This antitumor action has memory to the attack again of tumour, confirms to have long-term anti tumor immune response to exist.In addition, without tumor growth, and exist and to resist the immunizing power that tumour is attacked again after the tumor cell inoculation mouse of transfection 4-1BBL.Research is also found, the specific CTL s that fusion rotein Ig-4-1BBL effectively induced tumor the same as the anti-4-1 bb antibody continues, treatment Mouse Liver colorectal carcinoma.

On the other hand, confirm in the research of therapeutic vaccine, the recombinant poxvirus of expressing 4-1BBL obviously strengthens the vaccine-induced mouse of HIV and produces take IFN-γ and reply specific C D8 as feature ⁺T cell response thisly acts on immunity and still can detect in rear 2 months.In the CEA-transgene mouse model, the 4-1BBL recombinant poxvirus obviously strengthens the therapeutic action of the recombinant poxvirus vaccine of expressing B7.1 and CEA, this effect and CEA specific C D4 ⁺And CD8 ⁺The t cell response level increases relevant.Report is arranged recently, can induce powerful cell response and anamnestic reaction with several costimulatory moleculeses that comprise 4-1BBL as the molecule adjuvant of DNA vaccination.Above result has all been showed the application prospect of 4-1BBL in human tumor and infectious diseases immunotherapy.

Prior art is directly extracted from histocyte the preparation method of 4-1BBL albumen, and yield is low, cost is high.Can not be widely used in vivo test or clinical.

(3) summary of the invention

In order to improve the yield of 4-1BBL albumen, reduce costs, provide a large amount of starting material for preparing preclinical test medication or diagnostic reagent, the cDNA that the invention provides a kind of employing 4-1BBL (TNFSF9, NP_033430.1) extracellular fragment albumen contains the method for the restructuring 4-1BBL albumen of 206 amino-acid residues (Arg 104-Glu 309) with the gene engineering method preparation.

The technical solution used in the present invention is:

A kind of preparation method of 4-1BBL albumen, described method comprises:

(1) get and contain 4-1BBL albumen Mouse lymphocyte, the conA stimulated in vitro is cultivated by the trizol cracking, and extracting obtains mRNA; The raw materials used mouse lymphocyte that contains 4-1BBL albumen for routine;

(2) the mRNA reverse transcription is obtained the cDNA of 4-1BBL extracellular fragment albumen;

(3) step (2) gained cDNA is carried out pcr amplification, obtain 4-1BBL albumen goal gene; The pcr amplification primer is: upstream primer: 5 '-CTC GAG AAA AGA AGAACC GAG CCA AGA CCT-3 ', downstream primer: 5 '-GAA TTC TTC CCATGG ATT ATC AGG-3 ';

(4) with step (3) PCR product with the directed ppic9 that inserts through same double digestion after Xho I and EcoR I double digestion, obtain recombinant plasmid ppic9-4-1BBL;

(5) recombinant plasmid ppic9-4-1BBL is converted into bacillus coli DH 5 alpha, through cultivating amplification, extracting and purifying, sequencing analysis, obtains the correct recombinant plasmid of order-checking;

(6) will check order correct recombinant plasmid transformed to the methanol yeast bacterium, the abduction delivering recombinant protein;

(7) the activation bacterial strain that will express recombinant protein is induced fermentation culture, and fermented liquid is centrifugal, gets the supernatant liquor separation and purification, obtains the 4-1BBL recombinant protein of purifying.

the nucleotide sequence of step (5) recombinant plasmid ppic9-4-1BBL is as follows: CTC, GAG, AAAAGA, AGA, ACC, GAG, CCA, AGA, CCT, GCA, TTG, ACC, ATT, ACT, ACT, TCTCCA, AAC, TTG, GGT, ACA, AGA, GAA, AAC, AAC, GCT, GAC, CAG, GTCACT, CCA, GTT, TCT, CAC, ATC, GGA, TGT, CCA, AAC, ACC, ACC, CAG, CAGGGA, TCT, CCT, GTT, TTT, GCT, AAG, TTG, TTG, GCC, AAA, AAC, CAG, GCTTCT, TTG, TGT, AAC, ACT, ACC, TTG, AAC, TGG, CAC, TCT, CAA, GAC, GGTGCC, GGT, TCT, TCT, TAC, TTG, TCT, CAG, GGT, TTG, AGA, TAC, GAG, GAGGAC, AAG, AAG, GAG, TTG, GTC, GTC, GAC, TCT, CCT, GGA, TTG, TAC, TACGTC, TTT, TTG, GAG, TTG, AAG, TTG, TCT, CCA, ACA, TTC, ACC, AAC, ACTGGA, CAC, AAG, GTT, CAG, GGA, TGG, GTT, TCT, TTG, GTT, TTG, CAG, GCTAAG, CCA, CAG, GTT, GAC, GAT, TTC, GAC, AAT, TTG, GCC, TTG, ACT, GTCGAA, TTG, TTC, CCT, TGC, TCT, ATG, GAA, AAC, AAA, TTG, GTC, GAC, AGATCT, TGG, TCT, CAA, TTG, TTG, TTG, TTG, AAG, GCC, GGA, CAC, AGA, TTGTCT, GTC, GGA, TTG, AGA, GCA, TAC, TTG, CAT, GGT, GCA, CAA, GAC, GCTTAC, AGA, GAC, TGG, GAG, TTG, TCT, TAC, CCT, AAC, ACC, ACT, TCT, TTCGGT, TTG, TTC, TTG, GTT, AAA, CCT, GAT, AAT, CCA, TGG, GAA, GAA, TTC.

The aminoacid sequence of obtained recombinant protein is as follows:

RTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE。

Use the 4-1BBL albumen that the inventive method obtains, it is a New Set with highly sensitive, high specific, the 4-1BBL protein immune animal Dispersal risk that uses this invention technology to obtain, can make diagnostic kit, be used for pathogenesis, diagnosis, differential diagnosis and the prognosis of above-mentioned immune correlated disease animal model and the observation of curative effect.The 4-1BBL albumen that uses the present invention to make finished product can be used for preparation in early stage and the research of clinical medicine, uses and curative effect as the adjuvant of vaccine.Above concrete application mode belongs to common practise for those of ordinary skills, therefore repeats no more.

Beneficial effect of the present invention is mainly reflected in: adopt the inventive method, can obtain in a large number 4-1BBL albumen, greatly improved the albumen yield, the 4-1BBL extracellular fragment albumen of acquisition can be applied to prepare to the test kit of 4-1BBL protein quantification or medicinal, has a extensive future.

(4) description of drawings

Fig. 1 is that SDS-PAGE and the Western blot of 4-1BBL extracellular region protein analyzes; A:SDS-PAGE, 1:protein marker; 2: the yeast supernatant that empty plasmid transforms; The yeast supernatant of 3:4-1BBL recombinant plasmid transformed, target protein are the approximately tripolymer of 70KD of molecular weight; B:Western blot;

The attach most importance to analysis (cpm) of the short T cell proliferation experiment of H4-1BBL of Fig. 2;

Fig. 3 is the IL-2 level (ng/L) of mouse T cell 48h culture supernatant;

Fig. 4 is the IFN-γ level (ng/L) of mouse T cell 48h culture supernatant.

(5) embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

The preparation of embodiment 1:4-1BBL albumen

(1) separate BALB/c mouse splenocyte (the aseptic BALB/c mouse spleen of getting, after grinding, crosses the glass nook closing member 150 eye mesh screens, obtain single cell suspension), the conA stimulated in vitro is cultivated (after the RPMI1640 washing, resuspended and adjust cell concn as 1 * 10 take the RPMI1640 (containing conA5 μ g/ml) that contains 10% foetal calf serum ⁶/ ml puts 37 ℃, 5% CO ₂Condition is cultivated 72h, and collecting cell is used for extracting total RNA.) by the trizol cracking (0.1ml cell/1ml trizol, 42 ℃ 30～40min), extracting obtains mRNA;

(2) mRNA reverse transcription (Superscript II reverse transcription test kit is available from Invitrogen company) is obtained the cDNA of 4-1BBL extracellular fragment albumen;

(3) carry out pcr amplification take step (2) gained cDNA as template, obtain 4-1BBL albumen goal gene;

The pcr amplification primer is:

Upstream primer: 5 '-CTC GAG AAA AGA AGA ACC GAG CCA AGA

CCT-3’

Downstream primer: 5 '-GAA TTC TTC CCA TGG ATT ATC AGG-3 ';

The PCR reaction solution forms (Buffer, dNTP, DNA Polymerase are available from Invitrogen company):

ddH ₂O 37.60μL

10×Buffer 5μL

dNTP mixture(2.5mM) 4μL

Upstream primer (20pmol/ μ L) 1 μ L

Downstream primer (20pmol/ μ L) 1 μ L

MgCl ₂(25mM) 0.5μL

CDNA template (approximately 10 ⁴Copy) 0.5 μ L

DNA Polymerase 0.4μL

Cumulative volume 50 μ L

The PCR reaction conditions:

Enter circulation after 95 ℃ of denaturing treatment 3min, 94 ℃ of 30S, 56 ℃ of 30S, 72 ℃ of 1min, 30 rear 72 ℃ of extension 7min of circulation;

(4) with step (3) PCR product directed ppic9 through same double digestion (NOVAGEN company buy) that inserts after with Xho I+EcoR I double digestion, obtain recombinant plasmid ppic9-4-1BBL;

(5) recombinant plasmid ppic9-4-1BBL is converted into bacillus coli DH 5 alpha, through cultivating amplification, extracting and purifying, also carrying out sequential analysis with full-automatic sequenator, obtains the correct recombinant plasmid (sequence is seen SEQ ID No.1) of order-checking;

(6) will check order correct recombinant plasmid transformed to methanol yeast bacterium (NOVAGEN company buy), cultivate (separation of supernatant after 2-YT substratum (containing kanamycin50 μ g/ml) cultivation in 96 hours under the condition of 28 ℃, separate each protein band with SDS-PAGE, and detect with Western Blot; Purified product is analyzed demonstration through 12% SDS-PAGE, approximately a special protein belt (Figure 1A) is arranged the 70kD vicinity at molecular weight, is indicated as the tripolymer of 4-1BBL self-assembling formation.Western blot result shows, this albumen mass-energy and goat-anti mouse 4-1BB L antibody are (available from R﹠amp; D company) hybridization forms the approximately monomer of 23KD of molecular weight after β ME processes, conform to expected results (Figure 1B).

(7) bacterial strain that will express recombinant protein is first drawn flat board (yeast SD solid medium) inoculation, and then amplification in liquid nutrient medium (yeast SD liquid nutrient medium), seed liquor 5% inoculum size by volume is linked in 2-YT substratum (containing kanamycin 50 μ g/ml), 28 ℃, 300r/min is more than high dissolved oxygen is cultured to 50OD.Induced 8 hours under high dissolved oxygen condition, put tank, centrifugal, get supernatant liquor and carry out the separation and purification operation:

A), supernatant liquor adopts the 0.45um membrane filtration, adds 1/2 volume H ₂O is diluted to electricity and leads＜5mS/cm, crosses anion-exchange column, and filler is Q XL (Amershame), balance liquid (50mMTris+2.5mMEDTA+0.1mMPMSF, pH7.6), elutriant are (50mMTris, 0.3MNaCl+2.5mMEDTA, pH7.6), one-step elution;

B), collect preparation of samples and cross hydrophobic chromatography, slowly stream adds 2 times of volumetric balance liquid (25mMPB+0.8M (NH ₄) ₂SO ₄, pH7.4) to sample, stir simultaneously, filler is Phenyl HP, and elutriant is 25mMPB, and (A liquid is balance liquid to pH7.4, B liquid is elutriant, carry out drip washing and wash-out with the A+B mixed solution), the step gradient elution accounts for 60% (this accounts for the volume ratio of A+B mixed solution as B liquid) take elutriant, 70% drip washing, 90%, 100% wash-out, changing liquid is H ₂O is wash-out again a time, all treats baseline stability before each wash-out.

C), SDS-PAGE as a result the display-object stripe size meet expection, in the end in single step purification, the foreign protein major part is worn in liquid at stream, wash-out can get purity sample preferably after drip washing.

D), collect 90% elution fraction and cross Sephadex G25 desalination, changing liquid is 25mMPB, pH7.4.

E), by immunoassay or biological activity determination 4-1BBL protein peak part, obtain the 4-1BBL extracellular fragment albumen (sequence is seen SEQ ID No.2) of purifying, freeze-drying is preserved.

The immunologic competence of embodiment 2:4-1BBL albumen

The 4-1BBL extracellular fragment albumen that adopts the inventive method to obtain, through stimulating the mouse T lymphocyte of vitro culture, can effectively strengthen T lymphopoiesis (enhancing rate relatively is 25.6%) and the cytokines such as secretion IL-2 (enhancing rate relatively is 53.2%), IFN-γ (enhancing rate relatively is 65.5%) of CD3 monoclonal antibody activation, show and have the collaborative hormesis of significant T cell (Fig. 2～Fig. 4).

Method: mouse boosting cell filters through nylon hair post and obtains the T lymphocyte, with 10 ⁵/ mL T cell is inoculated in 96 orifice plates, set up 1 PBS control group and 3 experimental group (4-1BBL, the anti-CD28 of anti-CD3+, the anti-CD28 of the anti-CD3+ of 4-1BBL+), wherein 4-1BBL concentration is 1 μ g/mL, anti-CD3 and anti-CD28 (available from ebioscience company) concentration is 10 μ g/mL, and each group is established 3 multiple holes.At 37 ℃, 5%CO ₂Cultivate 48h under condition, the collecting cell supernatant liquor detects respectively IL-2 and IFN-γ concentration with ELISA test kit (available from Diaclone company).Similarity condition is cultivated the T cell warp of 72h ³H-TdR method (reagent is available from Sigma company) detects T cell proliferation index (cpm).The data that in figure are three independent experiments represent with " means standard deviation ".

SEQUENCE LISTING

＜110〉Hangzhou Pedagogic University

＜120〉a kind of preparation method of 4-1BBL albumen

<130>

<160> 4

<170> PatentIn version 3.4

<210> 1

<211> 636

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 1

ctcgagaaaa gaagaaccga gccaagacct gcattgacca ttactacttc tccaaacttg 60

ggtacaagag aaaacaacgc tgaccaggtc actccagttt ctcacatcgg atgtccaaac 120

accacccagc agggatctcc tgtttttgct aagttgttgg ccaaaaacca ggcttctttg 180

tgtaacacta ccttgaactg gcactctcaa gacggtgccg gttcttctta cttgtctcag 240

ggtttgagat acgaggagga caagaaggag ttggtcgtcg actctcctgg attgtactac 300

gtctttttgg agttgaagtt gtctccaaca ttcaccaaca ctggacacaa ggttcaggga 360

tgggtttctt tggttttgca ggctaagcca caggttgacg atttcgacaa tttggccttg 420

actgtcgaat tgttcccttg ctctatggaa aacaaattgg tcgacagatc ttggtctcaa 480

ttgttgttgt tgaaggccgg acacagattg tctgtcggat tgagagcata cttgcatggt 540

gcacaagacg cttacagaga ctgggagttg tcttacccta acaccacttc tttcggtttg 600

ttcttggtta aacctgataa tccatgggaa gaattc 636

<210> 2

<211> 206

<212> PRT

<213> Unknown

<220>

＜223〉artificial sequence

<400> 2

Arg Thr Glu Pro Arg Pro Ala Leu Thr Ile Thr Thr Ser Pro Asn Leu

1 5 10 15

Gly Thr Arg Glu Asn Asn Ala Asp Gln Val Thr Pro Val Ser His Ile

20 25 30

Gly Cys Pro Asn Thr Thr Gln Gln Gly Ser Pro Val Phe Ala Lys Leu

35 40 45

Leu Ala Lys Asn Gln Ala Ser Leu Cys Asn Thr Thr Leu Asn Trp His

50 55 60

Ser Gln Asp Gly Ala Gly Ser Ser Tyr Leu Ser Gln Gly Leu Arg Tyr

65 70 75 80

Glu Glu Asp Lys Lys Glu Leu Val Val Asp Ser Pro Gly Leu Tyr Tyr

85 90 95

Val Phe Leu Glu Leu Lys Leu Ser Pro Thr Phe Thr Asn Thr Gly His

100 105 110

Lys Val Gln Gly Trp Val Ser Leu Val Leu Gln Ala Lys Pro Gln Val

115 120 125

Asp Asp Phe Asp Asn Leu Ala Leu Thr Val Glu Leu Phe Pro Cys Ser

130 135 140

Met Glu Asn Lys Leu Val Asp Arg Ser Trp Ser Gln Leu Leu Leu Leu

145 150 155 160

Lys Ala Gly His Arg Leu Ser Val Gly Leu Arg Ala Tyr Leu His Gly

165 170 175

Ala Gln Asp Ala Tyr Arg Asp Trp Glu Leu Ser Tyr Pro Asn Thr Thr

180 185 190

Ser Phe Gly Leu Phe Leu Val Lys Pro Asp Asn Pro Trp Glu

195 200 205

<210> 3

<211> 30

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 3

ctcgagaaaa gaagaaccga gccaagacct 30

<210> 4

<211> 24

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 4

gaattcttcc catggattat cagg 24

Claims

1. the preparation method of a 4-1BBL albumen, described method comprises:

(1) get the mouse lymphocyte that contains 4-1BBL albumen, the conA stimulated in vitro is cultivated by the trizol cracking, and extracting obtains mRNA;

(3) step (2) gained cDNA is carried out pcr amplification, obtain 4-1BBL albumen goal gene; The pcr amplification primer is: upstream primer: 5 '-CTC GAG AAA AGA AGA ACC GAG CCA AGA CCT-3 ', downstream primer: 5 '-GAA TTC TTC CCA TGG ATT ATC AGG-3 ';

(5) recombinant plasmid ppic9-4-1BBL is converted into bacillus coli DH 5 alpha,, through cultivating amplification, extracting and purifying, sequencing analysis, obtains the correct recombinant plasmid of order-checking; The recombinant plasmid ppic9-4-1BBL sequence is as follows: CTC GAG AAA AGA AGA ACC GAG CCA AGA CCT GCA TTG ACC ATT ACT ACT TCT CCA AAC TTG GGT ACA AGA GAA AAC AAC GCT GAC CAG GTC ACT CCA GTT TCT CAC ATC GGA TGT CCA AAC ACC ACC CAG CAG GGA TCT CCT GTT TTT GCT AAG TTG TTG GCC AAA AAC CAG GCT TCT TTG TGT AAC ACT ACC TTG AAC TGG CAC TCT CAA GAC GGT GCC GGT TCT TCT TAC TTG TCT CAG GGT TTG AGA TAC GAG GAG GAC AAG AAG GAG TTG GTC GTC GAC TCT CCT GGA TTG TAC TAC GTC TTT TTG GAG TTG AAG TTG TCT CCA ACA TTC ACC AAC ACT GGA CAC AAG GTT CAG GGA TGG GTT TCT TTG GTT TTG CAG GCT AAG CCA CAG GTT GAC GAT TTC GAC AAT TTG GCC TTG ACT GTC GAA TTG TTC CCT TGC TCT ATG GAA AAC AAA TTG GTC GAC AGA TCT TGG TCT CAA TTG TTG TTG TTG AAG GCC GGA CAC AGA TTG TCT GTC GGA TTG AGA GCA TAC TTG CAT GGT GCA CAA GAC GCT TAC AGA GAC TGG GAG TTG TCT TAC CCT AAC ACC ACT TCT TTC GGT TTG TTC TTG GTT AAA CCT GAT AAT CCA TGG GAA GAA TTC；