CN1869226A - Perch agglutination gene sequence - Google Patents
Perch agglutination gene sequence Download PDFInfo
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- CN1869226A CN1869226A CN 200610035461 CN200610035461A CN1869226A CN 1869226 A CN1869226 A CN 1869226A CN 200610035461 CN200610035461 CN 200610035461 CN 200610035461 A CN200610035461 A CN 200610035461A CN 1869226 A CN1869226 A CN 1869226A
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Abstract
The invention relates guarding area design annex primer of CDS sequence. Distilling total RNA from spleen perch, using PCR method to expand and combining RACE technology, the full expression sequence 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 gene field in the molecular biology, particularly about the gene of perch agglutination.
Background technology
Lectin is that the energy aggegation cell or the precipitation in a kind of non-immunity source contains sugared macromolecular protein or glycoprotein, generally has sugared binding specificity, can combine with the sugar of glycoprotein or glycolipid, its function relates to cell growth, differentiation, growth and immunity etc., particularly at the external cell of aggegation, participate in engulfing, packing, solidify, aspects such as hemostasis and trauma repair play an important role.
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.Nonspecific defense mechanism is played an important role in hydrocoles is protected from infection, and potential nonspecific defense mechanism can be had an effect when microorganism is invaded, can more effectively remove, degrade pathogenic micro-organism and other objectionable impurities.Studies show that in a large number hydrocoles non-specific immunity system plays a greater role than the specific immunity system in himself resistant effect.
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 the gene of perch agglutination in conjunction with the RACE technology with the conserved regions design of the CDS sequence of the gene of nearly source species lectin.The nucleotide sequence of this gene 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.) lectin 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 260bp.Adopting β-second eyeball phosphoramidite chemical method to carry out DNA after the design of primers synthesizes.
It is synthetic that the acid amides chemical method carries out DNA.
Primer sequence is:
F:5’GCAHATCCACATAGYCAACTC?3’
R:5’CGAAYCTCCGCCATCG?3’
4, agglutinin gene pcr amplification, clone
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 the mensuration of the gene of perch agglutination
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 perch agglutination dna homolog sequence.Comparison result is:
Sequences?producing?significant?alignments: (Bits) Value
gi|78191594|gb|ABB29992.1|FBP32II?precursor[Morone?chrysoos]
474 3e-132
gi|78191592|gb|ABB29991.1|FBP32II?precursor[Morone?saxatilis]
474 3e-132
gi|78191604|gb|ABB29997.1|FBP32[Morone?saxatilis]
451 2e-125
gi|78191588|gb|ABB29989.1|FBP32?precursor[Morone?saxatitis]
451 2e-125
gi|78191590|gb|ABB29990.1|FBP32?precursor[Morone?chrysops]
449 5e-125
gi|78191607|gb|ABB29999.1|FBP32[Morone?chrysops]
428 1e-118
The acquisition of advantage of the present invention: Ben Jiyin not only is the effect of researching fish lectin in inflammatory reaction, for the genesis mechanism of further inquiring into the fish inflammatory reaction lays the foundation, and can obtain the purifying protein of biologically active by gene order, 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.) lectin 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 260bp.Adopting β-second eyeball phosphoramidite chemical method to carry out DNA after the design of primers synthesizes.
Primer sequence is:
F:5’GCAHATCCACATAGYCAACTC?3’
R:5’CGAAYCTCCGCCATCG?3’
4. the clone of agglutinin gene cDNA complete sequence
With the degenerate primer of the conserved regions design of nearly source species agglutinin gene CDS sequence, the amplified fragments size is about 260bp.
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 dNTP 2 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 the mensuration of perch agglutination 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 perch agglutination dna homolog sequence.Comparison result:
Sequences?producing?significant?alignments: (Bits) Value
gi|78191594|gb|ABB29992.1|FBP32II?precursor[Morone?chrysops]
474 3e-132
gi|78191592|gb|ABB29991.1|FBP32II?precursor[Morone?saxatilis]
474 3e-132
gi|78191604|gb|ABB29997.1|FBP32[Morone?saxatilis]
451 2e-125
gi|78191588|gb|ABB29989.1|FBP32?precursor[Morone?saxatilis]
451 2e-125
gi|78191590|gb|ABB29990.1|FBP32?precursor[Morone?chrysops]
449 5e-125
gi|78191607|gb|ABB29999.1|FBP32[Morone?chrysops]
428 1e-118
Sequence table
<110〉Nanhai Aquatic Inst., Chinese Aquatic Scientific Research Inst
<120〉Lateolabrax agglutinin gene order
<160>2
<210>1
<211>1304
<212>RNA
<213〉spend perch (Lateolabrax Japonicus)
<220>
<221>3’UTP
<222>(973)...(1304)
<220>
<221>5’UTP
<222>(1)...(39)
<220>
<221>CDS
<222>(40)...(972)
<220>
<221>PolyA?site
<222>(1283)...(1304)
<220>
<221>PolyA?signal
<222>(1263)...(1267)
<400>1
acattgagtc?atccgtctcc?aggaagaaag?cagtccaga?atg?atg?aga?ctc?agt 54
Met?Met?Arg?Leu?Ser
1 5
gtt?ttc?ctt?gtg?ctc?ctc?ctc?tcg?gag?acg?tgc?tca?gct?tcc?act?tat 102
Val?Phe?Leu?Val?Leu?Leu?Leu?Ser?Glu?Thr?Cys?Ser?Ala?Ser?Thr?Tyr
6 12 18
gaa?aat?gtg?gcc?ttg?cgt?gga?aaa?gcg?acc?cag?tcg?agc?cgt?tat?ttg 150
Glu?Asn?Val?Ala?Leu?Arg?Gly?Lys?Ala?Thr?Gln?Ser?Ser?Arg?Tyr?Leu
22 28 34
cat?gcg?ttt?gga?ggt?gcc?tac?aat?gct?att?gat?gga?aac?aga?gag?tct 198
His?Ala?Phe?Gly?Gly?Ala?Tyr?Asn?Ala?Ile?Asp?Gly?Asn?Arg?Glu?Ser
38 44 50
cac?ttc?cac?tct?gga?tca?tgc?acc?cac?tct?gct?gaa?gag?acc?acc?ccc 246
His?Phe?His?Ser?Gly?Ser?Cys?Thr?His?Ser?Ala?Glu?Glu?Thr?Asn?Pro
54 60 66
tgg?tgg?aga?gtg?gac?ctg?ctg?gac?tcc?tat?gtc?gtc?acc?tcc?atc?acc 294
Trp?Trp?Arg?Val?Asp?Leu?Leu?Asp?Ser?Tyr?Val?Val?Thr?Ser?Ile?Thr
70 76 82
atc?acc?acc?aga?ggg?gac?tgc?tgt?gaa?caa?agg?atc?agc?ggg?ctg?cag 342
Ieu?Thr?Asn?Arg?Gly?Asp?Cys?Cys?Glu?Gln?Arg?Ile?Ser?Gly?Leu?Glu
86 92 98
atc?cac?ata?ggc?aac?tct?tta?aag?gac?aac?ggt?gcc?agt?aac?cca?atg 390
Ile?His?Ile?Gly?Asn?Ser?Leu?Lys?Asp?Asn?Gly?Ala?Ser?Asn?Pro?Met
102 108 114
gtt?ggc?aca?atc?tct?gaa?att?ggt?gca?gct?aag?tcg?ttc?tct?ctg?cct 438
Val?Gly?Thr?Ile?Ser?Glu?Ile?Gly?Ala?Ala?Lys?Ser?Phe?Ser?Leu?Pro
118 124 130
ttc?acc?gat?cgt?gtg?gag?gga?cgc?tat?gtg?act?ttg?gtt?ctg?cct?ggt 486
Phe?Thr?Asp?Arg?Val?Glu?Gly?Arg?Tyr?Val?Thr?Leu?Val?Leu?Pro?Gly
134 140 146
tca?gga?aag?tac?ctc?aca?ctc?tgc?gaa?gtg?gag?gtc?tat?ggg?tac?cgt 534
Ser?Gly?Lys?Tyr?Leu?Thr?Leu?Cys?Glu?Val?Glu?Val?Tyr?Gly?Tyr?Arg
150 156 162
gcc?cca?act?gga?gag?aac?ctg?gcc?atc?caa?gga?aaa?gcc?aca?cag?tcc 582
Ala?Pro?Thr?Gly?Glu?Asn?Leu?Ala?Ile?Glu?Gly?Lys?Ala?Thr?Gln?Ser
166 172 178
tca?ttg?ttt?caa?ttt?ggc?act?gca?tat?aat?gcc?att?gac?ggg?aat?cat 630
Ser?Leu?Phe?Glu?Phe?Gly?Thr?Ala?Tyr?Asn?Ala?Ile?Asp?Gly?Asn?His
182 188 194
gcc?agc?aag?tgg?gaa?gac?ggc?tcc?tgt?agt?cac?aca?ata?aac?aac?gtc 678
Ala?Ser?Lys?Trp?Glu?Asp?Gly?Ser?Cys?Ser?His?Thr?Ile?Asn?Asn?Val
198 204 210
aac?ccc?tgg?tgg?cga?ttg?gat?ctg?ggc?aaa?acc?cat?aaa?gtg?ttt?tct 726
Asn?Pro?Trp?Trp?Arg?Leu?Asp?Leu?Gly?Lys?Thr?His?Lys?Val?Phe?Ser
214 220 226
gtt?aag?ata?acc?aac?aca?gat?gaa?aac?cca?gaa?cga?ctc?gat?gga?gcg 774
Val?Lys?Ile?Thr?Asn?Thr?Asp?Glu?Asn?Pro?Glu?Arg?Leu?Asp?Gly?Ala
230 236 242
gag?att?cga?atc?gga?gat?tct?ctt?gaa?aca?atg?gca?aca?cac?ata?cca 822
Glu?Ile?Arg?Ile?Gly?Asp?Ser?Leu?Glu?Thr?Met?Ala?Thr?Thr?Ile?Pro
246 252 258
cat?gtg?ctg?tgg?atc?aca?aat?atc?ccc?ggc?ggt?gct?gtt?gaa?ttc?cag 870
His?Val?Leu?Trp?Ile?Thr?Asn?Ile?Pro?Gly?Gly?Ala?Val?Glu?Phe?Gln
262 268 274
tgt?aac?ggg?atg?gac?ggc?cgc?tac?gtt?acc?gta?gtc?atc?cca?gga?aga 918
Cys?Asn?Gly?Met?Asp?Gly?Arg?Tyr?Val?Thr?Val?Val?Ile?Pro?Gly?Arg
278 284 290
gaa?gag?ttc?ctg?aca?ctt?tgt?gag?gtg?gag?gtg?tat?ggc?tcc?aga?ctg 966
Glu?Glu?Phe?Leu?Thr?Leu?Cys?Glu?Val?Glu?Val?Tyr?Gly?Ser?Arg?Leu
294 300 306
gattag 972
Asp
310
gtctgaagaa?gttcaaaatg?cataagaaca?tttggctttt?actaactact?catacaactg 1032
gaattacaac?ttaagcttca?atatagtccg?aaaaaaagca?tccaaaatca?ttcctcgtga 1092
atcctagtat?gacagtaatt?gcattgtggg?atttttttac?ggaactattt?agtgcataaa 1152
tataatcttt?agccatgttt?tgatttctaa?tgcttttgtt?gtattttctt?ttttaatact 1212
aaacttttgt?tgtactgtaa?ccaacgtctt?cagctgaaaa?actgaaatac?aataaaatca 1272
ttgcaaaacc?caaaaaaaaa?aaaaaaaaaa?aa
<210>2
<211>310
<212>PRT
<213〉spend perch (Lateolabrax Japonicus)
<400>2
Met?Met?Arg?Leu?Ser?Val?Phe?Leu?Val?Leu?Leu?Leu?Ser?Glu?Thr?Cys
1 7 13
Ser?Ala?Ser?Thr?Tyr?Glu?Asn?Val?Ala?Leu?Arg?Gly?Lys?Ala?Thr?Gln
17 23 29
Ser?Ser?Arg?Tyr?Leu?His?Ala?Phe?Gly?Gly?Ala?Tyr?Asn?Ala?Ile?Asp
33 39 45
Gly?Asn?Arg?Glu?Ser?His?Phe?His?Ser?Gly?Ser?Cys?Thr?His?Ser?Ala
49 55 61
Glu?Glu?Thr?Asn?Pro?Trp?Trp?Arg?Val?Asp?Leu?Leu?Asp?Ser?Tyr?Val
65 71 77
Val?Thr?Ser?Ile?Thr?Ieu?Thr?Asn?Arg?Gly?Asp?Cys?Cys?Glu?Gln?Arg
81 87 93
Ile?Ser?Gly?Leu?Glu?le?His?Ile?Gly?Asn?Ser?Leu?Lys?Asp?Asn?Gly
97 103 109
Ala?Ser?Asn?Pro?Met?Val?Gly?Thr?Ile?Ser?Glu?Ile?Gly?Ala?Ala?Lys
113 119 125
Ser?Phe?Ser?Leu?Pro?Phe?Thr?Asp?Arg?Val?Glu?Gly?Arg?Tyr?Val?Thr
129 135 141
Leu?Val?Leu?Pro?Gly?Ser?Gly?Lys?Tyr?Leu?Thr?Leu?Cys?Glu?Val?Glu
145 151 157
Val?Tyr?Gly?Tyr?Arg?Ala?Pro?Thr?Gly?Glu?Asn?Leu?Ala?Ile?Glu?Gly
161 167 173
Lys?Ala?Thr?Gln?Ser?Ser?Leu?Phe?Glu?Phe?Gly?Thr?Ala?Tyr?Asn?Ala
177 183 189
Ile?Asp?Gly?Asn?His?Ala?Ser?Lys?Trp?Glu?Asp?Gly?Ser?Cys?Ser?His
193 199 205
Thr?Ile?Asn?Asn?Val?Asn?Pro?Trp?Trp?Arg?Leu?Asp?Leu?Gly?Lys?Thr
209 215 221
His?Lys?Val?Phe?Ser?Val?Lys?Ile?Thr?Asn?Thr?Asp?Glu?Asn?Pro?Glu
225 231 237
Arg?Leu?Asp?Gly?Ala?Glu?Ile?Arg?Ile?Gly?Asp?Ser?Leu?Glu?Thr?Met
241 247 253
Ala?Thr?Thr?Ile?Pro?His?Val?Leu?Trp?Ile?Thr?Asn?Ile?Pro?Gly?Gly
257 263 269
Ala?Val?Glu?Phe?Gln?Cys?Asn?Gly?Met?Asp?Gly?Arg?Tyr?Val?Thr?Val
273 279 285
Val?Ile?Pro?Gly?Arg?Glu?Glu?Phe?Leu?Thr?Leu?Cys?Glu?Val?Glu?Val
289 295 301
Tyr?Gly?Ser?Arg?Leu?Asp
305 310
Claims (2)
1, a kind of perch agglutination gene, its nucleotide sequence is as described in the SEQ ID NO.1 in the sequence table.
2, a kind of perch agglutination gene, its aminoacid sequence is as described in the SEQ ID NO.2 in the sequence table.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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CN 200610035461 CN1869226A (en) | 2006-05-15 | 2006-05-15 | Perch agglutination gene sequence |
CN 200710088881 CN101063131A (en) | 2006-05-15 | 2007-04-04 | Lateolabrax agglutinin gene order |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 200610035461 CN1869226A (en) | 2006-05-15 | 2006-05-15 | Perch agglutination gene sequence |
Publications (1)
Publication Number | Publication Date |
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CN1869226A true CN1869226A (en) | 2006-11-29 |
Family
ID=37443005
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN 200610035461 Pending CN1869226A (en) | 2006-05-15 | 2006-05-15 | Perch agglutination gene sequence |
Country Status (1)
Country | Link |
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CN (1) | CN1869226A (en) |
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2006
- 2006-05-15 CN CN 200610035461 patent/CN1869226A/en active Pending
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