CN100427599C - ALBSIN/IL-18MAT hybridization cytokine - Google Patents

ALBSIN/IL-18MAT hybridization cytokine Download PDF

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Publication number
CN100427599C
CN100427599C CNB2003101159820A CN200310115982A CN100427599C CN 100427599 C CN100427599 C CN 100427599C CN B2003101159820 A CNB2003101159820 A CN B2003101159820A CN 200310115982 A CN200310115982 A CN 200310115982A CN 100427599 C CN100427599 C CN 100427599C
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gat
aaa
gaa
att
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CNB2003101159820A
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Chinese (zh)
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CN1635120A (en
Inventor
谭岩
刘力华
方艳秋
段秀梅
许淑芬
姜艳芳
宋艳
刘玲丽
时阳
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First Hospital Jinlin University
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First Hospital Jinlin University
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Abstract

ALBsin is a signal peptide of human serum albumins, and IL-18MAT is a mature peptide of human IL-18. The present invention obtains the chimeric molecules of the ALBSIN and IL-18MAT with a gene restitution method which comprises the steps: amplifying ALBSIN sequences from human genome DNA, obtaining IL-18MAT sequences from human IL-18 prokaryotic expression plasmids, obtaining the antisense strand of an ALBSIN coded area and an IL-18MAT positive strand by the asymmetric PCR technology, producing hybrid coded sequences with ALBSIN and 18MAT via overlaying extending PGR(SOG-PCR), orientationally inserting pcDNA3, and constructing the eukaryotic expression plasmids of hybrid IL-18. The sequence result shows that the expression cassette of the recombinant plasmids is composed of two designed target sequences without frame shift. The expression plasmids can transfect 293, Vero and 7721 cells, and express human IL-18 with bioactivity.

Description

ALB SIN/ IL-18 MATThe hybrid cell factor
Technical field:
The invention belongs to biological field, particularly relate to a kind of reorganization and production method of IL-18 gene.
Background technology:
IL-18 is a monokine, and two kinds of existence forms are arranged: i.e. precursor (ProIL-18) and mature peptide, the 1-35 amino-acid residue of ProIL-18 is a leader.In its secretory cell, 35 asparagicacid residues of caspase-1 enzyme identification, enzyme is cut the peptide bond that this amino-acid residue constitutes, and forms ripe IL-18 justacrine and goes out born of the same parents, the performance biological action.Bibliographical information IL-18 can be used as the immunological adjuvant of tumor vaccine, may be applied to the biotherapy of cancer.Because lack caspas-1 in the tumour cell mostly, therefore, the tumour knurl seedling of transfection IL-18 is difficult to play predictive role.
Technology contents:
At IL-18 MATAdd ALB before the sequence SINSequence, IL-18 MATDirectly be secreted into outside the born of the same parents by the signal peptide approach, i.e. the IL-18 at tumor immune response is induced in secretion MATThe hybrid cell factor.
Nucleotide/amino acid fragment the sequence of the present invention's reorganization is:
cccgcaagctt?atg?aag?tgg?gta?acc?ttt?att?tcc?ctt?ctt?ttt?ctc?ttt
M K W V T F I S L L F L F
agc?tcg?gct?tat?tcc?tac?ttt?ggc?aag?ctt?gaa?tct?aaa?tta?tca?gtc
S S A Y S Y F G K L E S K L S V
ata?aga?aat?ttg?aat?gac?caa?gtt?ctc?ttc?att?gac?caa?gga?aat?cgg
I R N L N D Q V L F I D Q G N R
cct?cta?ttt?gaa?gat?atg?act?gat?tct?gac?tgt?aga?gat?aat?gca
P L F E D M T D S D C R D N A
ccc?cgg?acc?ata?ttt?att?ata?agt?atg?tat?aaa?gat?agc?cag?cct?aga
P R T I F I I S M Y K D S Q P R
ggt?atg?gct?gta?act?atc?tct?gtg?aag?tgt?gag?aaa?att?tca?act?ctc
G M A V T I S V K C E K I S T L
tcc?tgt?gag?aac?aaa?att?att?tcc?ttt?aag?gaa?atg?aat?cct?cct?gat
S C E N K I I S F K E M N P P D
aac?atc?aag?gat?aca?aaa?agt?gac?atc?ata?ttc?ttt?cag?aga?agt?gtc
N I K D T K S D I I F F Q R S V
cca?gga?cat?gat?aat?aag?atg?caa?ttt?gaa?tct?tca?tca?tac?gaa?gga
P G H D N K M Q F E S S S Y E G
tac?ttt?cta?gct?tgt?gaa?aaa?gag?aga?gac?ctt?ttt?aaa?ctc?att?ttg
Y F L A C E K E R D L F K L I L
aaa?aaa?gag?gat?gaa?ttg?ggg?gat?aga?tct?ata?atg?ttc?act?gtt?caa
K K E D E L G D R S I M F T V Q
aac?gaa?gac?tga?gggccctat
N E D stop
ALB of the present invention SIN/ IL-18 MATThe preparation method of the hybrid cell factor is the genomic dna amplification ALB from the people SINSequence; From the human il-18 eukaryon expression plasmid, obtain IL-18 again MATSequence then, obtains ALB by the asymmetric pcr technology SINThe antisense strand of coding region and IL-18 MATPeptide normal chain, last, adopt overlapping extension PCR (SOE-PCR) to produce and have ALB SINWith IL-18 MATThe hybridization encoding sequence, behind the product double digestion with plasmid pCDNA 3Connect the expression plasmid and the order-checking that make up hybridization IL-18.
Characteristics of the present invention and positively effect are at IL-18 MATAdd ALB before the sequence SINSequence, can make the secretion of cell IL-18 not be subjected to the restriction of caspas-1, but go out born of the same parents, make IL-18 when the antineoplastic immunoadjuvant function of performance by the Secretory Pathway of signal peptide, no longer adhere rigidly to and the cell that can express caspas-1, also improved the secretory volume of IL-18 simultaneously.System of elisa assay proof recombinant plasmid transfection tumor cell can measure high-level human il-18 and express.
Embodiment:
One, the reorganization of IL-18
1, the PCR design of primers is with synthetic: design 2 pairs of primers altogether.
Clone's primer is right: 1. 5 '-CCCGC AAGCTTATGAAGTGGGTAACCTT-3 '
(containing HindIII restriction enzyme site and initiator codon)
②5’-ATT? GGGCCC?CTAGTCTTCGTTTTGAACA-3’
(containing ApaI restriction enzyme site and terminator codon)
Hybridized primer is right: be used for two target sequence splicings
③5’- CTTATTCCTACTTTGGCA?AGCTTGAA-3’
④5’- TGCCAAAGTAGGAATAAG?CCGAGCTAAAGA-3’
Underscore is two kinds of primers, 5 ' end complementary sequences.
2, the extraction of genomic dna
Extract genomic dna by human peripheral blood single nucleus cell routinely.Purity and concentration adopt spectrophotometer method to measure.DNA concentration is 328.5 μ g/ml.
3, PGR amplification
(1) symmetrical PGR:
With the genomic dna is template, adds primer 1. 4., amplification ALB SINSequence; Add dNTP2 1, Tag polysaccharase 2.5U, 10 * Tag enzyme reaction buffer solution 5l, Mg in the above-mentioned reaction system again 2+(25mM/L) 9 μ l, add distilled water to 50l.The cyclic amplification program is: 94 ℃ of 45s of sex change, 57 ℃ of 15s of renaturation, 72 ℃ of 20s of extension, 72 ℃ of 5min are fully extended in totally 30 circulations.Sepharose, electrophoresis reclaim with the DNA purification kit after identifying the PCR product.
With the human il-18 recombinant expression plasmid is template, adds primer 2. 3., amplification IL-18 MATSequence adds dNTP3 1, Tag polysaccharase 2.5U, 10 * Tag enzyme reaction buffer solution 5l, Mg again in the above-mentioned reaction system 2+(25mM/L) 5.5 μ l, add distilled water to 50l.The cyclic amplification program is: 94 ℃ of 45s of sex change, 57 ℃ of 15s of renaturation, 72 ℃ of 45s of extension, 72 ℃ of 5min are fully extended in totally 30 circulations.Sepharose, electrophoresis reclaim with the DNA purification kit after identifying the PCR product.
(2) asymmetric pcr (Asymmetric PCR):
Template is 2 a μ l symmetry PCR product, and uses single primer 1. or 4. to carry out asymmetric pcr.The cyclic amplification program is: 94 ℃ of 45s of sex change, 57 ℃ of 15s of renaturation, 72 ℃ of 60s of extension, 72 ℃ of 5min are fully extended in totally 20 circulations.Product is identified and is adopted the continuous polyacrylamide gel electrophoresis of non-sex change, gel strength 8%.
(3)SOE-PCR：
ALB SINSymmetrical PCR product and IL-18 MATThe asymmetric pcr product with 1: 100 mixed, as the template of SOE-PCR, add primer 1. 2..Add 10 * PCR buffer (no Mg in the above-mentioned reaction system again 2+) 5 μ l, d NTP 3 μ l, Mg 2+(25mM/L) 10 μ l, TaqEx enzyme 0.5 μ l.The round-robin condition is first: 94 ℃ of 45s of sex change; 37 ℃ of 10min anneal; Extend 72 ℃ of 2min so that overlapping extension.Then 94 ℃ of 45s of sex change; 57 ℃ of 15s anneal; Do 30 circulations under the condition of 72 ℃ of 60s of extension.
Two, make up eukaryon expression plasmid pcDNA 3-IL-18 Hyh
1, double digestion reaction:
The IL-18 recombination and the linear carrier pcDNA that prepare the toughness end with Hind III and ApaI respectively 3
2, the connection of dna fragmentation:
IL-18 recombination and linear carrier pcDNA in the reaction system 3Volume ratio be 1: 15.16 ℃, connect 12-16 hour.
3, the evaluation of hybridization IL-18
(1) PCR identifies:
With pcDNA 3-IL-18 HyhBe template, respectively with primer 1. 4., primer 2. 3., 2. 1. primer be three groups of PCR.And set up two contrasts, and be template with genomic dna and human il-18 recombinant expression plasmid respectively, corresponding primer is for 1. 4. and 2. 3..Agarose electrophoresis is identified.
(2) recombinant plasmid order-checking, (ABI PRISM TM377XL DNA sequencer).Qualification result shows: pcDNA 3-IL-18 HyhInsertion sequence be 548bp.Contain ALB SINWith IL-18 MATSequence, and do not have frameshit.Collection of illustrative plates is as follows:
cccgcaagctt atg?aag?tgg?gta?acc?ttt?att?tcc?ctt?ctt?ttt?ctc?ttt
M K W V T F I S L L F L
F
agc?tcg?gct?tat?tcc?tac?ttt?ggc?aag?ctt?gaa?tct?aaa?tta?tca
gtc
S S A Y S Y F G K L E S K L
S V
ata?aga?aat?ttg?aat?gac?caa?gtt?ctc?ttc?att?gac?caa?gga?aat?cgg
I R N L N D Q V L F I D Q G
N R
cct?cta?ttt?gaa?gat?atg?act?gat?tct?gac?tgt?aga?gat?aat?gca
P L F E D M T D S D C R D
N A?ccc?cgg?acc?ata?ttt?att?ata?agt?atg?tat?aaa?gat?agc?cag
cct?aga
P R T I F I I S M Y K D S Q
P R
ggt?atg?gct?gta?act?atc?tct?gtg?aag?tgt?gag?aaa?att?tca?act?ctc
G M A V T I S V K C E K I S
T L
tcc?tgt?gag?aac?aaa?att?att?tcc?ttt?aag?gaa?atg?aat?cct?cct?gat
S C E N K I I S F K E M N P
P D
aac?atc?aag?gat?aca?aaa?agt?gac?atc?ata?ttc?ttt?cag?aga?agt?gtc
N I K D T K S D I I F F Q R
S V
cca?gga?cat?gat?aat?aag?atg?caa?ttt?gaa?tct?tca?tca?tac?gaa?gga
P G H D N K M Q F E S S S Y
E G
tac?ttt?cta?gct?tgt?gaa?aaa?gag?aga?gac?ctt?ttt?aaa?ctc?att?ttg
Y F L A C E K E R D L F K L
I L
aaa?aaa?gag?gat?gaa?ttg?ggg?gat?aga?tct?ata?atg?ttc?act?gtt?caa
K K E D E L G D R S I M F T
V Q
aac?gaa?gac?tga gggccctat
N E D stop
Three, the activity identification of eukaryon expression plasmid
1, the transfection of eukaryon expression plasmid
(1) 5 * 10 5293, Vero or 7721 cells are inoculated in the 35cm culture dish respectively, 37 ℃, 5%CO 2Following 24 hours of condition, make the cell attachment growth.Be resuspended in 2ml serum-free 1640 before transfection next day.
(2) 10 μ g recombinant plasmid dnas are added among the 2 μ l lipofectamine, add serum-free 1,640 200 μ l, and room temperature 45 minutes is injected cell cultures, transfection 24hrs after adding 19% serum, 1640800 μ l.
2, human il-18 detects: 100 μ l culture supernatant are added on IL-18 ELISA test kit, routine operation, OD 490The IL-18 content of expression culture supernatant, this hybridization IL-18 clone's transfectional cell destination gene expression and secretory volume are cloned apparently higher than wild-type IL-18.
Meaning of the present invention is: (1) IL-18 plays important immunoadjuvant function in the process of inducing specific T cellullar immunologic response; Tumour knurl seedling transfection hybridization IL-18 clone activates killer T cell at tumour by producing high-level IL-18.(2) gene vaccine of structure specific for tumour antigen inserts the hybridization IL-18 sequence of the present invention's foundation and does coexpression, may promote antigen-specific helper cell propagation, thereby promote tumor rejection.

Claims (2)

1, a kind of ALB SIN/ IL-18 MATThe hybrid cell factor is characterized in that: its aminoacid sequence is:
M K W V T F I S L L F L FS S A Y S Y F G K L E S K L S VI R N L N D Q V L F I D Q G N RP L F E D M T D S D C R D N AP R T I F I I S M Y K D S Q P RG M A V T I S V K C E K I S T LS C E N K I I S F K E M N P P DN I K D T K S D I I F F Q R S VP G H D N K M Q F E S S S Y E GY F L A C E K E R D L F K L I LK K E D E L G D R S I M F T V QN E D,：atg aag tgggta acc ttt att tcc ctt ctt ttt ctc ttt agc tcg gct tat tcc tacttt ggc aag ctt gaa tct aaa tta tca gtc ata aga aat ttg aat gaccaa gtt ctc ttc att gac caa gga aat cgg cct cta ttt gaa gat atgact gat tct gac tgt aga gat aat gca ccc cgg acc ata ttt att ataagt atg tat aaa gat agc cag cct aga ggt atg gct gta act atc tctgtg aag tgt gag aaa att tca act ctc tcc tgt gag aac aaa att atttcc ttt aag gaa atg aat cct cct gat aac atc aag gat aca aaa agtgac atc ata ttc ttt cag aga agt gtc cca gga cat gat aat aag atgcaa ttt gaa tct tca tca tac gaa gga tac ttt cta gct tgt gaa aaagag aga gac ctt ttt aaa ctc att ttg aaa aaa gag gat gaa ttg ggggat aga tct ata atg ttc act gtt caa aac gaa gac tga gggccctat。
2, a kind of ALB SIN/ I L-18 MATThe preparation method of the hybrid cell factor is characterized in that: from people's genomic dna amplification ALB SINSequence; From the human il-18 eukaryon expression plasmid, obtain IL-18 again MATSequence then, obtains ALB by the asymmetric pcr technology SINThe antisense strand of coding region and IL-18 MATThe normal chain of the coding region of peptide, last, adopt overlapping extension PCR to produce and have ALB SINWith IL-18 MATThe hybridization encoding sequence, behind the product double digestion with plasmid pCDNA 3Connect the expression plasmid that makes up hybridization IL-18.
CNB2003101159820A 2003-12-26 2003-12-26 ALBSIN/IL-18MAT hybridization cytokine Expired - Fee Related CN100427599C (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090312196A1 (en) * 2008-06-13 2009-12-17 Codexis, Inc. Method of synthesizing polynucleotide variants
CN108341853B (en) * 2017-01-22 2022-06-21 中国科学院化学研究所 Human serum albumin specificity recognition polypeptide and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029242A2 (en) * 1999-10-21 2001-04-26 Monsanto Company Post-translational modification of recombinant proteins produced in plants
CN1357622A (en) * 2001-11-02 2002-07-10 武汉大学 Human interleukin-12 recombinant insect virus strain and its prepn
WO2002079415A2 (en) * 2001-03-30 2002-10-10 Lexigen Pharmaceuticals Corp. Reducing the immunogenicity of fusion proteins

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029242A2 (en) * 1999-10-21 2001-04-26 Monsanto Company Post-translational modification of recombinant proteins produced in plants
WO2001029242A3 (en) * 1999-10-21 2002-02-21 Monsanto Co Post-translational modification of recombinant proteins produced in plants
WO2002079415A2 (en) * 2001-03-30 2002-10-10 Lexigen Pharmaceuticals Corp. Reducing the immunogenicity of fusion proteins
CN1357622A (en) * 2001-11-02 2002-07-10 武汉大学 Human interleukin-12 recombinant insect virus strain and its prepn

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
人白细胞介素18 的基因克隆及其在大肠杆菌中的表达. 赵春艳等.中国生物制品学杂志，第16卷第1期. 2003 *
白细胞介素-18 的研究进展. 韩明勇等.国外医学肿瘤学分册，第30卷第4期. 2003 *

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