CN1869229A - Perch interlenkin 8 gene sequence - Google Patents
Perch interlenkin 8 gene sequence Download PDFInfo
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- CN1869229A CN1869229A CN 200610035460 CN200610035460A CN1869229A CN 1869229 A CN1869229 A CN 1869229A CN 200610035460 CN200610035460 CN 200610035460 CN 200610035460 A CN200610035460 A CN 200610035460A CN 1869229 A CN1869229 A CN 1869229A
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- primer
- perch
- interleukin
- sequence
- glu
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Abstract
The invention relates to conservation area design annex primer of CDS sequence of IL8 genes. Distilling total RNA from spleen perch, using PCR method to expand and combining RACE technology, the full expression sequence of interleukin 8 (IL-8) would be cloned to perch agglutinin. It lays a foundation for researching fish inflammatory reaction. It would gain pure albumen with bioactivity to supply new theory foundation for new type disease against.
Description
Technical field
The present invention relates to the gene field in the molecular biology, particularly about flower perch interleukin 8 gene order.
Background technology
Interleukin 8 (interleukin 8 IL-8) gene claims to bite the neutrophil activation factor or granulocyte chemokine again, belongs to C-X-C type CF, is the CF of finding the earliest.It is produced behind inflammatory stimulus by various kinds of cell such as monokaryon-scavenger cell, vascular endothelial cell, epithelial cell and mastocyte, ability with chemotactic and activation neutrophilic granulocyte, comprise that attracting to bite neutrophil leucocyte moves to inflammation part, and induce them to take off particle, discharge N,O-Diacetylmuramidase, start the respiratory metabolism outburst, increase the multiple functions such as expression of cell surface adhesion molecule, in inflammation generation and evolution important effect being arranged, is a kind of important immune disease-resistance gene.Along with the development of mariculture industry, the disease problem is on the rise at present, therefore self starts with from the fish body, and improving fish body immunizing power is the trend of current disease control.So far also less to the research of flower perch immunogene, especially the research to IL-8 also belongs to blank.
Summary of the invention
The objective of the invention is by degenerate primer, and with the amplification of PCR method, be cloned into the full expressed sequence of colored perch interleukin 8 (IL-8) gene in conjunction with the RACE technology with the conserved regions design of the CDS sequence of nearly source species IL8 gene.Its Nucleotide is as described in the SEQ ID NO.1 in the sequence table; Its aminoacid sequence is as described in the SEQ ID NO.2 in the sequence table.
Purpose of the present invention realizes by following technical measures:
1, the extraction of total RNA
Dissect the fish body and take out spleen, use Trizol to carry out homogenate, extract the total RNA of flower perch according to operation instruction.2, cDNA first chain is synthetic
The total RNA of flower perch mixes with reverse transcription primer (oligo-dT joint primer), carries out reverse transcription.
3, design of primers foundation, primer synthetic method
From GenBank, download nearly source species (as people, ox, rainbow trout, lefteye flounder, carp etc.) IL8 homologous gene CDS sequence, utilize Clustal W software to carry out the multisequencing comparison, determine conserved regions, according to conserved regions sequences Design degenerate primer, the amplified fragments size is about 180bp.Adopting β-second eyeball phosphoramidite chemical method to carry out DNA after the design of primers synthesizes.
Primer sequence is:
F：5’TGCCRCTGCATHGARAC?3’
R：5’ACTTGTTVATGACYHTCTTVACCCA?3’
4, the clone of IL-8 gene cDNA complete sequence
As template, carry out pcr amplification with degenerate primer with the above-mentioned synthetic first chain cDNA, institute's amplification PCR products detects with 1.2% agarose gel electrophoresis, and purifying reclaims the purpose product from gel.Then with the PCR product cloning of purifying in the pMD-18T carrier, transformed into escherichia coli JM-109 competent cell, the picking positive colony extracts plasmid DNA.After degenerated primer PCR detects, will have the segmental plasmid DNA of insertion and carry out two-way order-checking with the M13 universal primer.
According to the cDNA fragment sequence that obtained design Auele Specific Primer, utilize the terminal rapid amplifying technology of cDNA (Rapid Amplification of cDNA ends, RACE) to 3 of goal gene ' and 5 ' end carry out pcr amplification.
In 3 ' RACE, utilize half-nest type (semi-nested) PCR method, at first carry out the PCR reaction by outside primer and joint primer, products therefrom utilization inboard and joint primer carry out pcr amplification again.
In 5 ' RACE, utilize terminal enzyme (DNA) and dCTP after the cDNA end adds poly (C) tail, with the cDNA behind the tailing as template, utilize outside Auele Specific Primer and OligodG to carry out the pcr amplification first time, gained PCR product utilizes inboard primer and OligodG to carry out the pcr amplification second time again.
Resulting PCR product is after carrying out separation detection on 1.2% the agarose gel electrophoresis, behind the PCR product purification, be cloned in the pMD-18T carrier, transformed into escherichia coli JM-109 competent cell, the picking positive colony, with the M13 primer plasmid DNA is checked order, order-checking institute calling sequence utilizes Clustal W software to splice with the sequence of degenerate primer amplification gained again.
5, to spending the mensuration of perch interleukin 8 (IL-8) gene
Resulting PCR product is after carrying out separation detection on 1.2% the agarose gel electrophoresis, behind the PCR product purification, be cloned in the pMD-18T carrier transformed into escherichia coli JM-109 competent cell, the picking positive colony checks order to plasmid DNA with 3730 sequenators.Sequencing result carries out homology with BLAST software to be measured, and is defined as flower perch IL-8 homologous sequence.Comparison result:
Score E
Sequences?producing?significant?alignments： (Bits) Value
gi|74095881|ref|NP?001027759.1|interleukin-8[Takifugu?rubri...
159 2e-38
gi|62901686|gb|AAY18807.1|interleukin?8[Acanthopagrus?schlege|
154 1e-36
gi|19911219|dbj|BAB86884.1|CXC?chemokine?[Paralichthys?olivaceu
146 3e-34
gi|15667642|gb|AAL05442.1|interleukine-8[Paralichthys?olivaceu
144 8e-34
gi|52547128|gb|AAU81660.1|interleukin-8[Gadus?morhua]
141 9e-33
gi|77621310|emb|CAD59734.2|interleukin-8[Gadus?morhua]
138 7e-32
gi|91701717|gb|ABE47600.1|interleukin-8[Cyprinus?carpio]
130 2e-29
gi|31087942|gb|AAP42829.1|interleukin?8X[Oncorhynchus?mykis...
126 2e-28
Advantage of the present invention: along with the development of mariculture industry, the disease problem is on the rise, and general antibiotic medicine very easily causes environmental pollution, therefore self starts with from the fish body, and improving fish body immunizing power is the trend of disease control.Therefore the acquisition of this gene, not only be the effect of researching fish IL-8 in inflammatory reaction, the genesis mechanism of further inquiring into the fish inflammatory reaction lays the foundation, and can obtain the purifying protein of biologically active by aminoacid sequence, provide fundamental basis for studying novel anti pathologic immunity adjuvant.
Embodiment
1. the extraction of total RNA
Get fresh and alive healthy flower perch (the about 400g of body weight) and in the laboratory, behind the temporarily foster 2d (about 24 ℃ of water temperature, air-pump inflating), inject lipopolysaccharides (LPS, 10 μ g/mL) 200 μ L from the thoracic cavity.After stimulating 6h, dissect the fish body and take out the about 100mg of spleen, (Gibco carries out homogenate in Japan), extracts total RNA according to the test kit operation instruction to put into 1mL Trizol respectively.
2.cDNA first chain is synthetic
Getting the total RNA 5 μ g of colored perch mixes with reverse transcription primer (oligo-dT joint primer) 1 μ L (10pmol/L), behind 70 ℃ of heating 5min, place on ice immediately, add 5 * buffer then, 2.5mmol/L dNTP mixed solution, Ribonuclease Inhibitor, M-MLV ThermoScript II, reaction system are 25 μ L.Reaction process is 42 ℃ of 60min, 70 ℃ of 15min, and it is standby to put into-80 ℃ of preservations at last.
3. design of primers foundation, primer synthetic method
From GenBank, download nearly source species (as people, ox, rainbow trout, lefteye flounder, carp etc.) IL8 homologous gene CDS sequence, utilize Clustal W software to carry out the multisequencing comparison, determine conserved regions, according to conserved regions sequences Design degenerate primer, the amplified fragments size is about 180bp.Adopting β-second eyeball phosphoramidite chemical method to carry out DNA after the design of primers synthesizes.
Primer sequence is:
F：5’TGCCRCTGCATHGARAC?3’
R：5’ACTTGTTVATGACYHTCTTVACCCA?3’
4.IL-8 the clone of gene cDNA complete sequence
With the degenerate primer of the conserved regions design of nearly source species IL8 gene C DS sequence, the amplified fragments size is about 180bp.
As template, carry out pcr amplification with degenerate primer with the above-mentioned synthetic first chain cDNA, reaction system is: 10xPCR reaction buffer 5 μ L, 25mmol/L MgCl
23 μ L, 2.5mmol/L dNTP2 μ L, each 2 μ L of 10nmol/L primer dTF and dTR, Taq enzyme 1.25U is supplemented to 50 μ L with PCR water with reaction system.Reaction conditions is: 1 circulation, 94 ℃ of sex change 5min; 35 circulations: 94 ℃ of sex change 45s, 56 ℃ of annealing 45s, 72 ℃ are extended 45s; 1 circulation, 72 ℃ are extended 10min; 4 ℃ of insulations.Institute's amplification PCR products detects with 1.2% agarose gel electrophoresis, and purifying reclaims the purpose product from gel.Then with the PCR product cloning of purifying in the pMD-18T carrier, transformed into escherichia coli JM-109 competent cell, the picking positive colony extracts plasmid DNA.After degenerated primer PCR detects, will have the segmental plasmid DNA of insertion and carry out two-way order-checking with the M13 universal primer.
According to the cDNA fragment sequence design Auele Specific Primer that has obtained.Utilize the terminal rapid amplifying technology of cDNA (Rapid Amplification of cDNA ends, RACE) to 3 of goal gene ' and 5 ' end carry out pcr amplification.
In 3 ' RACE, utilize half-nest type (semi-nested) PCR method, at first carry out the PCR reaction by outside primer and joint primer, products therefrom is got 1 μ L as template after diluting 50 times, utilizes inboard and joint primer to carry out pcr amplification again.
In 5 ' RACE, utilize terminal enzyme (DNA) and dCTP after the cDNA end adds poly (C) tail, with the cDNA behind the tailing as template, utilize outside Auele Specific Primer and OligodG to carry out the pcr amplification first time, gained PCR product is got 1 μ L as template, utilizes inboard primer and OligodG to carry out the pcr amplification second time again.
Resulting PCR product is after carrying out separation detection on 1.2% the agarose gel electrophoresis, behind the PCR product purification, be cloned in the pMD-18T carrier, transformed into escherichia coli JM-109 competent cell, the picking positive colony, with the M13 primer plasmid DNA is checked order, order-checking institute calling sequence utilizes Clustal W software to splice with the sequence of degenerate primer amplification gained again.
5. to spending the mensuration of perch interleukin 8 (IL-8) gene
Resulting PCR product is after carrying out separation detection on 1.2% the agarose gel electrophoresis, behind the PCR product purification, be cloned in the pMD-18T carrier transformed into escherichia coli JM-109 competent cell, the picking positive colony checks order to plasmid DNA with 3730 sequenators.Sequencing result carries out homology with BLAST software to be measured, and is defined as flower perch IL-8 homologous sequence.Comparison result:
Score E
Sequences?producing?significant?alignments： (Bits) Value
gi|74095881|ref|NP?001027759.1|interleukin-8[Takifugu?rubri...
159 2e-38
gi|62901686|gb|AAY18807.1|interleukin?8[Acanthopagrus?schlegel
154 1e-36
gi|19911219|dbj|BAB86884.1|CXC?chemokine[Paralichthys?olivaceu
146 3e-34
gi|15667642|gb|AAL05442.1|interleukine-8[Paralichthys?olivaceu
144 8e-34
gi|52547128|gb|AAU81660.1|interleukin-8[Gadus?morhua]
141 9e-33
gi|77621310|emb|CAD59734.2|interleukin?8[Gadus?morhua]
138 7e-32
gi|91701717|gb|ABE47600.1|interleukin?8[Cypr?inus?carpio]
130 2e-29
gi|31087942|gb|AAP42829.1|interleukin?8X[Oncorhynchus?mykis...
126 2e-28
Sequence table
＜110〉Nanhai Aquatic Inst., Chinese Aquatic Scientific Research Inst
＜120〉spend perch interleukin 8 gene order
<160>2
<210>1
<211>803
<212>RNA
＜213〉spend perch (Lateolabrax Japonicus)
<220>
<221>3’UTP
<222>(460)…(803)
<220>
<221>5’UTP
<222>(1)...(159)
<220>
<221>CDS
<222>(160)...(459)
<220>
<221>PolyA?site
<222>(788)...(803)
<400>1
ccaagaaaag?gaaaagtaga?gagagtgaac?tcagtgaaag?aataaggaat?acagaagaat 60
aaaaaacttc?tttactttta?tacaggcttc?atcagacggc?tttctgaagg?gcattcatat 120
ccttagtgat?aatttgttgc?aaaatttgta?aaaggcaaa?atg?aag?agc?agc?aga 174
Met?Lys?Ser?Ser?Arg
1 5
gtg?att?gtc?acc?tct?act?gtg?gtg?ctc?ctg?gct?ttc?ttg?gcc?atc?act 222
Val?Ile?Val?Thr?Ser?Thr?Val?Val?Leu?Leu?Ala?Phe?Leu?Ala?Ile?Thr
6 12 18
gaa?ggg?atg?agt?ctg?aga?agc?ctt?gga?gtg?gag?ctg?cac?tgc?cgc?tgc 270
Glu?Gly?Met?Ser?Leu?Arg?Ser?Leu?Gly?Val?Glu?Leu?His?Cys?Arg?Cys
22 28 34
att?gag?acg?gag?agc?aaa?ccc?atc?ggc?cgc?cac?att?gag?aag?gtg?gag 318
Ile?Glu?Thr?Glu?Ser?Lys?Pro?Ile?Gly?Arg?His?Ile?Glu?Lys?Val?Glu
38 44 50
ctg?att?cct?gcc?aac?tcc?cac?tgc?gag?gag?act?gag?ctc?att?gcc?act 366
Leu?Ile?Pro?Ala?Asn?Ser?His?Cys?Glu?Glu?Thr?Glu?Ile?Ile?Ala?Thr
54 60 66
ctg?aag?aag?aca?ggc?caa?gaa?gtt?tgc?ctg?gac?ccg?gag?gct?ccc?tgg 414
Leu?Lys?Lys?Thr?Gly?Gln?Glu?Val?Cys?Leu?Asp?Pro?Glu?Ala?Pro?Trp
70 76 82
gtc?aag?aaa?gtc?atg?aac?aag?ttc?ctg?tcc?aac?aga?aca?ccc?tga 459
Val?Lys?Lys?Val?Met?Asn?Lys?Phe?Leu?Ser?Asn?Arg?Thr?Pro
86 92 98
acagatcggg?agagatgtgt?ttcatgagtc?tgaacaattt?caaagtacta?aaaagtattt 519
atttgatagt?catcacactc?aatttaatac?aatcaactgt?caatttgatc?aactgttgaa 579
aatgacaaca?gaatttacca?agtaaggtta?tgtttgtatc?aaacatgtgt?gttaacaagc 639
ctgtgcttgt?ttgtatgtca?cttgtgtgtg?ttactgtgta?ttcttatcta?taacttattt 699
gcttaaaata?tttattgata?tatttatgat?gtaaatgtca?attgtttagc?tctgctgaca 759
accaattgat?ttattaaaaa?agtttaccta?aaaaaaaaaaa?aaa 803
<210>2
<211>99
<212>PRT
＜213〉spend perch (Lateolabrax Japonicus)
<400>2
Met?Lys?Ser?Ser?Arg?Val?Ile?Val?Thr?Ser?Thr?Val?Val?Leu?Leu
1 7 13
Ala?Phe?Leu?Ala?Ile?Thr?Glu?Gly?Met?Ser?Leu?Arg?Ser?Leu?Gly
16 22 28
Val?Glu?Leu?His?Cys?Arg?Cys?Ile?Glu?Thr?Glu?Ser?Lys?Pro?Ile
31 37 43
Gly?Art?His?Ile?Glu?Lys?Val?Glu?Leu?Ile?Pro?Ala?Asn?Ser?His
46 52 58
Cys?Glu?Glu?Thr?Glu?Ile?Ile?Ala?Thr?Leu?Lys?Lys?Thr?Gly?Gln
61 67 73
Glu?Val?Cys?Leu?Asp?Pro?Glu?Ala?Pro?Trp?Val?Lys?Lys?Val?Met
76 82 88
Asn?Lys?Phe?Leu?Ser?Asn?Arg?Tht?Pro
91 97
Claims (2)
1, a kind of colored perch interleukin 8 gene, its nucleotide sequence is as described in the SEQ ID NO.1.
2, a kind of colored perch interleukin 8 gene, its aminoacid sequence is as described in the SEQ ID NO.2.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610035460 CN1869229A (en) | 2006-05-15 | 2006-05-15 | Perch interlenkin 8 gene sequence |
| CN 200710088884 CN101063132A (en) | 2006-05-15 | 2007-04-05 | Lateolabrax interleukins 8 gene order |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610035460 CN1869229A (en) | 2006-05-15 | 2006-05-15 | Perch interlenkin 8 gene sequence |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1869229A true CN1869229A (en) | 2006-11-29 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610035460 Pending CN1869229A (en) | 2006-05-15 | 2006-05-15 | Perch interlenkin 8 gene sequence |
Country Status (1)
| Country | Link |
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| CN (1) | CN1869229A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108424916A (en) * | 2018-03-29 | 2018-08-21 | 中国水产科学研究院南海水产研究所 | Flower perch interleukins IL-12p40 genes and application thereof |
-
2006
- 2006-05-15 CN CN 200610035460 patent/CN1869229A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108424916A (en) * | 2018-03-29 | 2018-08-21 | 中国水产科学研究院南海水产研究所 | Flower perch interleukins IL-12p40 genes and application thereof |
| CN108424916B (en) * | 2018-03-29 | 2021-04-30 | 中国水产科学研究院南海水产研究所 | Lateolabrax japonicus interleukin IL-12p40 gene and application thereof |
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