CN1511842A

CN1511842A - Rockfish insulin-like growth factor I gene and carrier containing said gene and recombined strain and its use

Info

Publication number: CN1511842A
Application number: CNA021497966A
Authority: CN
Inventors: 李文笙; 石锋涛; 林浩然
Original assignee: National Sun Yat Sen University
Current assignee: Sun Yat Sen University; National Sun Yat Sen University
Priority date: 2002-12-31
Filing date: 2002-12-31
Publication date: 2004-07-14

Abstract

The present invention discloses a kind of rockfish insulin-like growth factor I gene. The gene is gene segment with rockfish insulin-like growth factor I gene full length cDNA sequence obtained with rockfish liver total RNA as template and through RT-PCR and RACE process. The present invention also constitutes carrier and recombinant strain containing the gene. The present invention makes it possible to obtain rockfish insulin-like growth factor I recombinant protein with stable source and low cost and to prepare fish growth promoter and additive suitable for sea water fishes.

Description

Cabrilla insulin-like growth factor I gene and contain carrier and the recombinant strain and the application thereof of this gene

Technical field

The invention belongs to gene engineering technology field, particularly a kind of cabrilla insulin-like growth factor I gene and contain the carrier and the recombinant strain of this gene, and this recombinant protein is fit to short fish growth promoter that seawater fish uses or the application in the additive in preparation.

Background technology

Insulin-like growth factor I (Insulin-like growth factor-I) is a kind of protein of being made up of 70 amino acid, because its structure is similar to proinsulin (proinsulin), so and Regular Insulin (insulin, INS), insulin like growth factor-1 I (IGF-II), Relaxin (relaxin) be called as the INS/IGF/relaxin family protein together, IGF-I has and regulates cellular metabolism, promotes cell growth, differentiation and division, suppresses necrocytosis, regulates the effect of various kinds of cell function.The growth enhancing effect of tethelin (GH) is mainly by IGF-I mediation, so IGF-I also once was known as somatomedin C (somatomedin C), and IGF-I plays an important role in postnatal growing.

Because IGF-I has important role in many-sides such as fish growth, breeding and osmotic pressure adjustings, external many scholars have set up transgenic animal model overexpression IGF-I gene, and the growth rate of IGF-I transgenic animal has showed the increase of slight (about 30%).

In recent years along with fish IGF-I research further deeply, no matter be bioactive detection of IGF-I and research, still set up the IGF-I radioimmunoassay, radioreceptor assay, or ELISA etc. needs q.s fish IGF-I albumen.Many scholars natural IGF-I albumen of once attempting to purify from blood plasma of fish or tissue is all come back after a vain attempt, and external now existing company begins scale operation gene recombination fish IGF-I albumen, but the IGF-I albumen of Epinephelus coioide is not arranged as yet.

Summary of the invention

The object of the present invention is to provide a kind of reorganization Epinephelus coioide insulin-like growth factor I gene;

Another object of the present invention is to provide the carrier that contains this gene, particularly cloning vector and expression vector;

Another object of the present invention is to provide above-mentioned carrier microorganism transformed bacterial strain;

Another object of the present invention is to provide above-mentioned recombinant protein to be fit to the short fish growth promoter of seawater fish use or the application in the additive in preparation.

Amino acid sequence analysis shows that Epinephelus coioide insulin-like growth factor I mature peptide is a former interior fragment of Epinephelus coioide insulin-like growth factor I, be equivalent to the Epinephelus coioide insulin-like growth factor I former the 45th to 112 amino acids sequences, be the B-C-A-D zone, totally 68 amino acid.Insulin-like growth factor I is former to produce this section through proteolytic ferment excision back in the course of processing, have the biological activity that promotes growth, breeding and adjusting osmotic pressure, does not have this activity and complete insulin-like growth factor I is former.

Epinephelus coioide insulin-like growth factor I full-length gene of the present invention is to be template with the cDNA that the reverse transcription of Epinephelus coioide liver total RNA obtains, obtain through PCR and the amplification of RACE method, it derives from the pairing full length gene fragment of insulin-like growth factor I structural domain on the Epinephelus coioide liver cDNA.

The IGF-I mature peptide gene order that the present invention is used to express Epinephelus coioide IGF-I recombinant protein is to be template with Epinephelus coioide insulin-like growth factor I full-length gene double-stranded DNA, obtain through the PCR method amplification, it derives from the B-C-A-D area relative gene fragment of Epinephelus coioide insulin-like growth factor I full-length gene.

IGF-I mature peptide gene order according to above-mentioned Epinephelus coioide IGF-I recombinant protein, it is the fragment of Epinephelus coioide insulin-like growth factor I full-length gene 394bp to 597bp (being equivalent to 45 to 112 amino acids) preferably, and its aminoacid sequence and coded aminoacid sequence thereof are shown in sequence table.

The present invention has also made up the carrier of the IGF-I mature peptide gene order that contains above-mentioned Epinephelus coioide IGF-I recombinant protein; The escherichia coli cloning carrier and the expression vector that particularly contain this gene.These construction of carrier are according to a conventional method, will by PCR method synthetic Epinephelus coioide insulin-like growth factor I gene through enzyme cut with separation and purification after, be linked between the corresponding restriction enzyme site of existing respective carrier, promptly be built into the required Epinephelus coioide insulin-like growth factor I expression carrier that contains.

The recombinant expression vector that the above-mentioned coli expression carrier that contains Epinephelus coioide insulin-like growth factor I gene preferably is built into by synthetic Epinephelus coioide insulin-like growth factor I gene of the present invention and coli expression carrier pRSET, called after pRSET-IGF-I.

The present invention has also made up the intestinal bacteria recombinant strain that can efficiently express Epinephelus coioide insulin-like growth factor I gene respectively with the above-mentioned corresponding expression vectors that contains Epinephelus coioide IGF-I gene.

Of the present inventionly above-mentionedly can express the bacterial strain that Epinephelus coioide insulin-like growth factor I intestinal bacteria recombinant bacterial strain preferably obtains by containing Epinephelus coioide insulin-like growth factor I expression carrier pRSET-IGF-I transformed into escherichia coli BL21, called after Escherichia coliBL21 (pRSET-IGF-I) is called for short Ecoli BL21 (pRSET-IGF-I).

Epinephelus coioide has great economic worth, and the present invention utilizes genetic engineering technique production reorganization Epinephelus coioide IGF-I albumen, and can be further as short fish growth promoter of preparation or additive.

Description of drawings

Fig. 1 and Fig. 2 are to be the gel electrophoresis analysis figure of the insulin-like growth factor I gene intermediate segment pcr amplification product of template with the cDNA that the reverse transcription of Epinephelus coioide liver total RNA obtains;

Fig. 3 is Epinephelus coioide insulin-like growth factor I gene a 3 ' RACE operational flowchart;

Fig. 4 is Epinephelus coioide insulin-like growth factor I gene 3 ' end fragment synthetic PCR gel electrophoresis analysis figure for the first time;

Fig. 5 is Epinephelus coioide insulin-like growth factor I gene 3 ' end fragment synthetic PCR gel electrophoresis analysis figure for the second time;

Fig. 6 is Epinephelus coioide insulin-like growth factor I gene 3 ' end fragment synthetic PCR gel electrophoresis analysis figure for the third time;

Fig. 7 is an Epinephelus coioide insulin-like growth factor I gene 5 ' RACE operational flowchart;

Fig. 8 is Epinephelus coioide insulin-like growth factor I gene 5 ' end fragment synthetic PCR gel electrophoresis analysis figure for the first time;

Fig. 9 is Epinephelus coioide insulin-like growth factor I gene 5 ' end fragment synthetic PCR gel electrophoresis analysis figure for the second time;

Wherein: 1. molecular weight standard; 2.PCR product; 3.pGEM-T-easy vector; 4.pGEM-T-IGF-I-clone/EcoRI.

Embodiment

The invention will be further described below in conjunction with accompanying drawing, but do not limit the present invention in any form.

Synthesizing of embodiment 1 Epinephelus coioide insulin-like growth factor I gene intermediate segment

Homology comparative result according to the cDNA sequence of the insulin-like growth factor Is of people who has delivered and various animals, to classify the design of primers template as with the cDNA total order of the nearest black porgy of Epinephelus coioide affinity, the nested degenerated primer of the synthetic cover of design has two upstream primers and a downstream primer.Article one, upstream primer (PF1) is from 20 bases of the 259th bit base [5 '-CCACACCCTCTCACT (A/G) is TGC (C/G/T)],

Second upstream primer (PF2) is from 23 bases of the 369th bit base

【5’-GTGGAGA(G/C)AG(A/G)GG(C/G/A)TTTTATTTC】，

Downstream primer (PR1) is from 20 bases of the 708th bit base

【5’-CG(A/G)CT(T/C)GAGTT(T/C)TTC(G/T)GATG】。

For the first time the cDNA that obtains with the reverse transcription of Epinephelus coioide liver total RNA of PCR is a template, and through PCR method former the 259th to 708 nucleotide sequence that increases, reaction conditions is: 95 ℃ of pre-sex change 5 minutes; Bottom is 3 circulations: 95 ℃ of sex change 30 seconds, and 80 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute; Last 72 ℃ were extended 7 minutes.For the second time PCR with the first time PCR product be template, through PCR method former the 369th to 708 nucleotide sequence that increases, reaction conditions is: 95 ℃ of pre-sex change 5 minutes; Bottom is 3 circulations: 95 ℃ of sex change 30 seconds, and 80 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute; Last 72 ℃ were extended 7 minutes.

The electrophoresis evaluation figure of twice PCR amplified production sees Fig. 1 and Fig. 2.From the visible pcr amplification for the first time of Fig. 1 the sequence of the about 450bp of one segment length, and increased the visible second time sequence of the about 340bp of a segment length of Fig. 2.

The amplified production of 340bp is connected to obtains recombinant plasmid pGEM-T-IGF-I-Clone on the pGEM-T-easy carrier.Recombinant plasmid pGEM-T-IGF-I-Clone is carried out sequencing with a pair of universal primer of pGEM-T-easy, the sequence of sequencing result and Genebank compares, and the result shows that clone's PCR product is the intermediate segment of Epinephelus coioide insulin-like growth factor I gene.

Synthesizing of embodiment 2 Epinephelus coioide insulin-like growth factor I genes 3 ' end fragment

Operation is undertaken by 3 ' RACE System for Rapid Amplification of cDNA EndsKit Protocol.According to the requirement of test kit, according to pulsating sequences Design in the middle of the IGF-I that checked order two upstream primer GSP and Nested GSP to carry out nested PCR.Article one, upstream primer (GSP) is 22 bases of middle pulsating sequence the 242nd bit base of IGF-I from having checked order

【5’-TAGGACAGCACAGCAGCCAGAC】，

Second upstream primer (Nested GSP) is from 22 bases of the 280th bit base

【5’-AGAGACCTTTACCTGGACATAG】。Idiographic flow is seen Fig. 3:

For the first time the cDNA that obtains with the reverse transcription of Epinephelus coioide liver total RNA of PCR is a template, and through PCR method former the 369th nucleotide sequence to 3 ' poly A end that increase, reaction conditions is: 95 ℃ of pre-sex change 5 minutes; Bottom is 3 circulations: 95 ℃ of sex change 30 seconds, and 80 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute; Last 72 ℃ were extended 7 minutes.For the second time PCR with the first time PCR product be template, through PCR method former the 611st nucleotide sequence to 3 ' poly A end that increase, reaction conditions is: 95 ℃ of pre-sex change 5 minutes; Bottom is 3 circulations: 95 ℃ of sex change 30 seconds, and 80 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute; Last 72 ℃ were extended 7 minutes.For the third time PCR with the second time PCR product be template, through PCR method former the 649th nucleotide sequence to 3 ' poly A end that increase, reaction conditions is: 95 ℃ of pre-sex change 5 minutes; Bottom is 3 circulations: 95 ℃ of sex change 30 seconds, and 80 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute; Last 72 ℃ were extended 7 minutes.

The electrophoresis evaluation figure of three pcr amplification products sees Fig. 4, Fig. 5 and Fig. 6.

The amplified production of 750bp and 250bp is connected to obtains recombinant plasmid pGEM-T-IGF-I-3 '-1 and pGEM-T-IGF-I-3 '-2 on the pGEM-T-easy carrier.The a pair of universal primer of recombinant plasmid pGEM-T-IGF-I-3 '-1 and pGEM-T-IGF-I-3 '-2 usefulness pGEM-T-easy is carried out sequencing, the sequence of sequencing result and Genebank compares, the result show clone's 250bp product be Epinephelus coioide insulin-like growth factor I 3 ' end group because of.

Synthesizing of embodiment 3 Epinephelus coioide insulin-like growth factor I gene 5 ' end fragments

Operation is undertaken by 5 ' RACE System for Rapid Amplification of cDNA EndsKit Protocol.According to the requirement of test kit, according to pulsating sequences Design in the middle of the IGF-I that checked order downstream primer G-GSP1 to carry out reverse transcription and two downstream primer S-GSP2 and Nested S-GSP to carry out nested PCR.Article one, downstream primer (G-GSP1) is 17 bases of middle pulsating sequence the 317th bit base of IGF-I from having checked order

【5’-CTTGAAGGATGAATGACTAT】，

Second downstream primer (S-GSP2) is from 19 bases of the 89th bit base

【5’-GAAGCAGCATTCGTCCACA】，

Article three, downstream primer (Nested S-GSP) is from 19 bases of the 44th bit base

【5’-GCCATAGCCTGTTGGTTTA】。

Idiographic flow is seen Fig. 7.

For the first time the cDNA that obtains with the reverse transcription of Epinephelus coioide liver total RNA of PCR is a template, and through PCR method former the 459th nucleotide sequence to 5 ' end that increase, reaction conditions is: 95 ℃ of pre-sex change 5 minutes; Bottom is 3 circulations: 95 ℃ of sex change 30 seconds, and 80 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute; Last 72 ℃ were extended 7 minutes.For the second time PCR with the first time PCR product be template, through PCR method former the 414th nucleotide sequence to 5 ' end that increase, reaction conditions is: 95 ℃ of pre-sex change 5 minutes; Bottom is 3 circulations: 95 ℃ of sex change 30 seconds, and 80 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute; Last 72 ℃ were extended 7 minutes.

The electrophoresis evaluation figure of secondary PCR amplified production sees Fig. 8 and Fig. 9.

The amplified production of 650bp and 400bp is connected to obtains recombinant plasmid pGEM-T-IGF-I-5 '-1 and pGEM-T-IGF-I-5 '-2 on the pGEM-T-easy carrier.The a pair of universal primer of recombinant plasmid pGEM-T-IGF-I-5 '-1 and pGEM-T-IGF-I-5 '-2 usefulness pGEM-T-easy is carried out sequencing, the sequence of sequencing result and Genebank compares, the result show clone's 650bp product be Epinephelus coioide insulin-like growth factor I 5 ' end group because of.

The splicing of embodiment 4 Epinephelus coioide insulin-like growth factor I full length gene sequences

The IGF-I coded slices, 5 ' RACE segment and 3 ' the RACE segment that have checked order are spliced, obtain the total length of the IGF-I gene of Epinephelus coioide, be 925bp, and begin from the initiation codon of 262bp, finish to its termination codon, infer its aminoacid sequence.The aminoacid sequence of IGF-I full length gene and supposition is seen sequence table.

Sequence table

＜110〉Zhongshan University

＜120〉cabrilla insulin-like growth factor I gene and contain carrier and the recombinant strain and the application thereof of this gene

<130>

<140>

<141>

<160>2

<170>PatentIn?Ver.2.1

<210>1

<211>925

<212>DNA

<220>

<221>CIDS

<222>(262)..(819)

<220>

<221>sig_peptide

<222>(262)..(393)

<220>

<221>polyA_signal

<222>(909)..(925)

<400>1

gggggggggg?ggaggcaaat?gctgccccag?ctgtttcctg?ttgaaaatgt?ctgtgtaatg?60

tagataaatg?tgagggattt?tctctctaaa?tccgtctcct?gttcgctaaa?tctcacttct?120

ccaaaacgag?cctgcgcaat?ggaacaaagt?cggaatattg?agatgtgaca?ttgcccgcat?180

ctcatcctct?ttctccccgt?tttttaatga?cttcaaacaa?gttcattttc?gccgggcttt?240

gtcttgcgga?gacccgtggg?g?atg?tct?agc?gct?ctt?tcc?ttt?cag?tgg?cat 291

Met?Ser?Ser?Ala?Leu?Ser?Phe?Gln?Trp?His

1 5 10

tta?tgt?gat?gtc?ttc?aag?agt?gcg?atg?tgt?tgt?atc?tcc?tgt?agccac 339

Leu?Cys?Asp?Val?Phe?Lys?Ser?Ala?Met?Cys?Cys?Ile?Ser?Cys?Ser?His

15 20 25

acc?ctc?tca?cta?ctg?ctg?tgc?gtc?ctc?acc?ctg?act?ccg?acg?gca?aca 387

Thr?Leu?Ser?Leu?Leu?Leu?Cys?Val?Leu?Thr?Leu?Thr?Pro?Thr?Ala?Thr

30 35 40

ggg?gcg?ggc?cca?gag?acc?ctg?tgc?ggg?gcg?gag?ctg?gtc?gac?acg?ctg 435

Gly?Ala?Gly?Pro?Glu?Thr?Leu?Cys?Gly?Ala?Glu?Leu?Val?Asp?Thr?Leu

45 50 55

cag?ttt?gtg?tgt?gga?gag?aga?ggc?ttt?tat?ttc?agt?aaa?cca?aca?ggc 483

Gln?Phe?Val?Cys?Gly?Glu?Arg?Gly?Phe?Tyr?Phe?Ser?Lys?Pro?Thr?Gly

60 65 70

tat?ggc?ccc?aat?gta?cgg?cgg?tca?cgt?ggc?att?gtg?gac?gaa?tgc?tgc 531

Tyr?Gly?Pro?Asn?Val?Arg?Arg?Ser?Arg?Gly?Ile?Val?Asp?Glu?Cys?Cys

75 80 85 90

ttc?caa?agc?tgt?gag?ctg?cgg?cgc?ctg?gag?atg?aac?tgt?gca?cct?gcc 579

Phe?Gln?Ser?Cys?Glu?Leu?Arg?Arg?Leu?Glu?Met?Asn?Cys?Ala?Pro?Ala

95 100 105

aag?act?agc?aag?gct?gct?cgc?tct?gtg?cgt?gca?cag?cgc?cac?aca?gac 627

Lys?Thr?Ser?Lys?Ala?Ala?Arg?Ser?Val?Arg?Ala?Gln?Arg?His?Thr?Asp

110 115 120

atg?ccg?aga?gca?ccc ~?gtt?agt?acc?gca?ggg?cac?aaa?gtg?gac?aaa 675

Met?Pro?Arg?Ala?Pro?L Val?Ser?Thr?Ala?Gly?His?Lys?Val?Asp?Lys

125 130 135

ggc?aca?gag?cgt?agg?aca?gca?cag?cag?cca?gac?aag?aca?aaa?aac?aag 723

Gly?Thr?Glu?Arg?Arg?Thr?Ala?Gln?Gln?Pro?Asp?Lys?Thr?Lys?Asn?Lys

140 145 150

aag?aga?cct?tta?cct?gga?cat?agt?cat?tca?tcc?ttc?aag?gaa?gtg?cat 771

Lys?Arg?Pro?Leu?Pro?Gly?His?Ser?His?Ser?Ser?Phe?Lys?Glu?Val?His

155 160 165 170

cag?aaa?aac?tca?agt?cga?ggc?aac?acg?ggg?ggc?aga?aat?tac?aga?atg 819

Gln?Lys?Asn?Ser?Ser?Arg?Gly?Asn?Thr?Gly?Gly?Arg?Asn?Tyr?Arg?Met

175 180 185

tagggaaggt?gcgaatggac?aaatgcccag?caacttggga?agagagaagg?gagtggcctt?879

acctggtacc?cctgtggaat?ggttcactgt?aaaaaaaaaa?aaaaaa 925

<210>2

<211>186

<212>PRT

<400>2

Met?Ser?Ser?Ala?Leu?Ser?Phe?Gln?Trp?His?Leu?Cys?Asp?Val?Phe?Lys

1 5 10 15

Ser?Ala?Met?Cys?Cys?Ile?Ser?Cys?Ser?His?Thr?Leu?Ser?Leu?Leu?Leu

20 25 30

Cys?Val?Leu?Thr?Leu?Thr?Pro?Thr?Ala?Thr?Gly?Ala?Gly?Pro?Glu?Thr

35 40 45

Leu?Cys?Gly?Ala?Glu?Leu?Val?Asp?Thr?Leu?Gln?Phe?Val?Cys?Gly?Glu

50 55 60

Arg?Gly?Phe?Tyr?Phe?Ser?Lys?Pro?Thr?Gly?Tyr?Gly?Pro?Asn?Val?Arg

65 70 75 80

Arg?Ser?Arg?Gly?Ile?Val?Asp?Glu?Cys?Cys?Phe?Gln?Ser?Cys?Glu?Leu

85 90 95

Arg?Arg?Leu?Glu?Met?Asn?Cys?Ala?Pro?Ala?Lys?Thr?Ser?Lys?Ala?Ala

100 105 110

Arg?Ser?Val?Arg?Ala?Gln?Arg?His?Thr?Asp?Met?Pro?Arg?Ala?Pro?Lys

115 120 125

Val?Ser?Thr?Ala?Gly?His?Lys?Val?Asp?Lys?Gly?Thr?Glu?Arg?Arg?Thr

130 135 140

Ala?Gln?Gln?Pro?Asp?Lys?Thr?Lys?Asn?Lys?Lys?Arg?Pro?Leu?Pro?Gly

145 150 155 160

His?Ser?His?Ser?Ser?Phe?Lys?Glu?Val?His?Gln?Lys?Asn?Ser?Ser?Arg

165 170 175

Gly?Asn?Thr?Gly?Gly?Arg?Ash?Tyr?Arg?Met

180 185

Claims

1, a kind of cabrilla insulin-like growth factor I gene, gene order and aminoacid sequence are shown in sequence table.

2, cabrilla insulin-like growth factor I gene as claimed in claim 1, it is characterized in that it is is template with the cDNA that the reverse transcription of cabrilla liver total RNA obtains, the gene fragment that obtains through PCR and the amplification of RACE method with cabrilla insulin-like growth factor I full length cDNA sequence.

3, a kind of cloning vector is characterized in that containing the described cabrilla insulin-like growth factor I of claim 1 gene.

4, cloning vector as claimed in claim 3 is characterized in that this carrier is escherichia coli cloning carrier pGEMT-IGF-I.

5, a kind of recombinant strain is characterized in that it being to change formed intestinal bacteria recombinant strain pGEMT-IGF-I-DH5 α among the intestinal bacteria E.coli.DH5 α over to by the described cloning vector pGEMT-IGF-I of claim 4.

6, a kind of expression vector is characterized in that containing the described cabrilla insulin-like growth factor I of claim 1 gene.

7, expression vector as claimed in claim 6 is characterized in that this carrier is coli expression carrier pRSET-IGF-I.

8, a kind of recombinant strain is characterized in that it being to change formed intestinal bacteria recombinant strain pRSET-IGF-I-BL21 in the e. coli bl21 (DE3) over to by the described expression vector pRSET-IGF-I of claim 7.

9, the cabrilla insulin-like growth factor I recombinant protein of claim 1 is fit to the short fish growth promoter of seawater fish use or the application in the additive in preparation.