CN103911338A

CN103911338A - Construction of engineering strain capable of highly expressing IGF-1 based on procaryotic codon preference

Info

Publication number: CN103911338A
Application number: CN201410118148.5A
Authority: CN
Inventors: 刘文洪; 叶志青; 王炜钢; 李俊峰; 张贝贝; 贾孟荻
Original assignee: Zhejiang Chinese Medicine University ZCMU
Current assignee: Zhejiang Chinese Medicine University ZCMU
Priority date: 2014-03-27
Filing date: 2014-03-27
Publication date: 2014-07-09

Abstract

The invention relates to construction of an engineering strain capable of highly expressing IGF-1 based on procaryotic codon preference. According to a procaryotic codon use frequency table, a full gene sequence and a human protogene sequence with different preferences are artificially synthesized, cloned to different expression vectors, and transformed to differently expressed colibacillus strains to construct 27 engineering strains. An engineering strain subjected to preference modification and capable of efficiently expressing fusion protein is obtained through PCR screening positive cloning, IPTG induced expression, SDS-Page electrophoresis and HPLC detection, and is named RPP2; through liquid chromatography detection, the expression amount of a target protein of RPP2 is 2.4 times the maximal expression amount of the constructed human protogene engineering strain without modification; if being applied to industrial production, the strain is capable of greatly improving industrial output and bringing prominent economic benefit for enterprises.

Description

Build the engineering bacteria of high expression level IGF-1 gene based on protokaryon codon preference

Technical field

The structure that the present invention relates to recombinant human insulin-like growth factor (hIGF-1) gene, belongs to biology field, specifically relates to a kind of engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference.

Background technology

Insulin-like growth factor I GF-1(insulin-like growth factors-1, IGF-1) be that a class had not only had the polypeptide that promotes cytodifferentiation and propagation but also have ILA, be a kind of activated protein peptide material that human body itself contains, IGF-1 is playing a very important role aspect hypoglycemic, reducing blood-fat, vasodilator, growth promotion, short cytodifferentiation, trauma repair, antitumor, cardiovascular disorder and anti-ageing waiting for a long time.In view of the extensive and important physiological function of IGF-1, its obstacle abnormal and system function synthetic and secretion can cause a lot of serious diseases, also has diabetes; Insuline resistance syndrome; Nanism; The multiple persistent ailment such as cardiovascular and cerebrovascular and nervous system disorders is also closely bound up with the physiological function of IGF-1, therefore utilizes gene engineering method to obtain the IGF-1 goods that have in a large number biologic activity, and its fundamental research and clinical application are had to profound significance.

But the most of experiment of production research of IGF-1 is all also in the laboratory small-scale stage, while being mainly IGF-1 production, output is few, and separation difficulty, yields poorly, expensive (approximately 100000 yuan/gram), and this causes the development and utilization of IGF-1 to be had a strong impact on.The output that how to expand IGF-1, reduces costs, and has become extremely urgent work.

Summary of the invention

The deficiency existing in order to overcome prior art, overcome the low problem of eukaryotic gene expression amount in prokaryotic hosts, the invention provides a kind of engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference, by designing multiple optimizing codon sequence, build different expression vectors, prepare multiple expression strain.

A kind of engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference, described engineering bacteria is can be at the genetic engineering bacterium of intracellular expression or excreting and expressing recombinant human insulin-like growth factor fusion proteins, described engineering bacteria is colibacillus engineering, and the aminoacid sequence of described fusion rotein is shown in Seq ID No:4.

Preferably, described engineering bacteria comprises the recombinant expression vector and the expression strain that contain described high expression level IGF-1 gene.

Preferably, the carrier that sets out of described recombinant expression vector is that its gene order is shown in Seq ID No:1 without the IGF-1 people source protogene sequence of optimizing.

Preferably, 1. described IGF-1 gene comprises complete synthesis IGF-1 Preference order after codon optimized, and its gene order is shown in Seq ID No:2.

Preferably, 2. described IGF-1 gene comprises complete synthesis IGF-1 Preference order after codon optimized, and its gene order is shown in Seq ID No:3.

Preferably, described recombinant expression vector comprises intracellular expression carrier and secretion expression carrier.

Preferably, described intracellular expression carrier be by IGF-1 Preference order 1. or IGF-1 Preference order 2. DNA molecular be inserted into the recombinant plasmid that the multiple clone site place of plasmid PGEX4T-1 and PET32a obtains, in described intracellular expression carrier, starting the promotor that described DNA transcribes is T7 promotor.

Preferably, described secretion expression carrier be by IGF-1 Preference order 1. or IGF-1 Preference order 2. DNA molecular be inserted into the recombinant plasmid that the multiple clone site place of plasmid PET22b obtains, in described secretion expression carrier, starting the promotor that described DNA transcribes is T7 promotor.

Preferably, described intestinal bacteria comprise Escherichia coli BL21 (DE3), Rosetta (DE3) and Origami (DE3).

Preferably, described expression strain comprises the engineering strain of a strain through Preference transformation, and 2., the RPP 2. expression amount of target protein is constructed 2.4 times without the high expression level amount of transformation people source protogene engineering bacteria to this project bacterial strain called after RPP.This project bacterial strain is the engineering strain of the efficient expressed fusion protein of energy.

Different plant species is inconsistent in the frequency of utilization of genetic codon, and the codon that in some eukaryotic gene, the frequency of occurrences is very high but seldom uses in prokaryotic gene.In the present invention, according to e. coli codon frequency of utilization table synthetic and cloned and be suitable for the gene at the IGF-1 of expression in escherichia coli, transcribe to increase expression amount by what improve goal gene mRNA, from improving in essence the expression of target protein.

The present invention is suitable for the gene at the IGF-1 of expression in escherichia coli according to protokaryon codon usage frequency table synthetic clone, goal gene (comprising through the IGF-1 gene of Preference transformation with without the former IGF-1 gene in people source of transforming) hereinafter described.

In the present invention, goal gene contains 243 bases, and this 243 base sequence can translate and contain 77 amino acid whose peptide chains.

The present invention designs 3 kinds of codon sequences of expressing IGF-1 altogether, builds 9 kinds of recombinant expression vectors and screens to obtain 27 strain engineering bacterias.

In the present invention the highest recombinant expression vector of IGF-1 target protein expression amount be PGEX4T-IGF-1 2., this expression vector starts the promotor that described DNA molecular transcribes and has T7 promotor, described goal gene is to be inserted into the recombinant plasmid that the multiple clone site place of plasmid PGEX4T-1 obtains, described multiple clone site is BamHI and EcoRI, the structure of this fusion plasmid as shown in Figure 1, construction step is: (1) uses BamHI and two kinds of restriction enzymes of EcoRI at 36 DEG C, and under 5h, difference double digestion expression plasmid PGEX4T-1 and recombinant plasmid PUC57-IGF-1 are 2.; (2) separate enzyme with 1.0% agarose gel electrophoresis and cut product, rubber tapping reclaim goal gene fragment and cut after linear plasmid, then with DNA sepharose reclaim purification kit from agarose, isolate goal gene fragment and be cut after linear plasmid; (3) object band and cut after linear plasmid in the ratio T of 3:1 ₄dNA ligase spends the night 16 DEG C of connections; (4) the Transfected Recombinant Plasmid Escherichia coli JM109 competent cell connecting, coating contains the LB flat board of penbritin, 37 DEG C of cultivations, 12h, 10 single bacterium colonies of picking detect positive colonies.

The present invention utilizes the GST label of described expression vector and goal gene to build the fusion rotein forming, and inserts enteropeptidase restriction enzyme site between GST label and goal gene, and the aminoacid sequence of described fusion rotein is shown in GST-IGF-1 aminoacid sequence in sequence table.

In invention, related goal gene is transcribed obtained RNA molecule, and the cloning vector that contains described goal gene, expression vector, preservation bacterial strain and expression strain also belong to protection scope of the present invention.

In intestinal bacteria, the IGF-1 gene of codon optimization provided by the present invention with without transformation the former IGF-1 gene in people source compared with, screening has obtained a strain through the efficiently engineering strain of expressed fusion protein of Preference transformation, this project bacterial strain called after RPP 2., through Liquid Detection RPP 2. the expression amount of target protein be constructed 2.4 times without the high expression level amount of transformation people source protogene engineering bacteria.

The present invention by the synthetic IGF-1 of e. coli codon Preference complete gene constructed go out the IGF-1 engineering bacteria of high expression level and the foundation of high-density culture technology thereof, for new approach is explored in the production of IGF-1, can effectively solve IGF-1 natural amount in application and production process low, the problems such as production cost is high, and product is impure.

The present invention is according to protokaryon codon usage frequency table, synthetic difference preference complete genome sequence and people source protogene sequence, and be cloned on different expression vectors, be then transformed into different expression in coli strains, build altogether 27 strain engineering bacterias.PCR screening positive clone, IPTG abduction delivering, SDS-Page electrophoresis and HPLC detect, screening has obtained a strain through the efficiently engineering strain of expressed fusion protein of Preference transformation, this project bacterial strain called after RPP 2., through Liquid Detection RPP 2. the expression amount of target protein be constructed 2.4 times without the high expression level amount of transformation people source protogene engineering bacteria, if this project bacterium for suitability for industrialized production, can greatly be improved industrial output value also for enterprise brings distinct economic.

Brief description of the drawings

Fig. 1 is that 2. PGEX4T-IGF-1 of the present invention builds schematic diagram;

Fig. 2 is that restriction enzyme mapping carries out agarose gel electrophoresis detection figure;

Fig. 3 is that PCR product carries out agarose gel electrophoresis figure;

Fig. 4 is the SDS-PAGE electrophorogram of each bacterial strain recombinant expression;

Fig. 5 is that final concentration is the IGF-1 standard model HPLC figure of 50ug/mL;

Fig. 6 is the IGF-1 people source raw sample HPLC figure of 2 times of dilutions;

Fig. 7 is the 2. sample HPLC figure of IGF-1 of 2 times of dilutions.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described, but protection scope of the present invention is not limited to this.

The experimental technique using in following embodiment if no special instructions, is ordinary method.

Goal gene (comprising IGF-1 Preference order and IGF-1 people's source sequence): synthesize and be implemented in PUC57 carrier by Shanghai biotechnology company limited; Expression vector PGEX4T-1 carrier, PET22b carrier and PET32 carrier: purchased from Addgene company; Intestinal bacteria (comprising Escherichia coli BL21 (DE3), Rosetta (DE3) and Origami (DE3)): provided by Zhejiang University's Life Science College.Other reagent: other reagent required in experiment provide by Biological Engineering College of Zhejiang University of Traditional Chinese Medicine genetically engineered laboratory.

The present invention not only provides and has planted the engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference, and the recombinant expression vector and the expression strain that contain IGF-1 gene are also provided simultaneously.

1, the codon optimized and full gene of insulin-like growth factor I GF-1 is synthetic

First, download IGF-1 human source gene sequence (insulin-like growth factors-1, IGF-1) at NCBI, according to e. coli codon frequency of utilization table design IGF-1 Preference sequence, thereby improve the expression level of IGF-1 in intestinal bacteria.Again by the synthetic IGF-1 people's source protogene sequence of Shanghai biotechnology company limited and codon optimized after IGF-1 gene order and be implemented on PUC57 cloned plasmids, be finally stored in DH5 α bacterial strain.

Design respectively IGF-1 preference and IGF-1 people source primer sequence with primer 5.0, more synthetic by Shanghai biotechnology company limited, primer sequence is in table 1.

Table 1

Primer title	Primer sequence (5'to3')	Purification process
			The former IGF-1 F in people source	cgtggatccgatgatgatgataaaggacc	HAP
The former IGF-1 R in people source	ctggaattctcattaagctgacttggca	HAP
			Preference IGF-1 F 1.	cgtggatccgatgatgatgataaaggtcc	HAP
Preference IGF-1 R 1.	ctggaattctcattacgcagatttcgcc	HAP
			Preference IGF-1 F 2.	cgtggatccgatgatgatgataaaggacc	HAP
Preference IGF-1 R 2.	ctggaattctcattacgcagatttcgcc	HAP

2, the structure of recombinant plasmid and qualification

By recombinant clone plasmid (comprise: recombinant plasmid PUC57-IGF-1 1., recombinant plasmid PUC57-IGF-1 2. and the former recombinant plasmid PUC57-IGF-1 in people source) and expression vector (comprising PGEX4T-1, PET22b and PET32a) use respectively BamHI and EcoRI(purchased from Fermentas company) double digestion.Enzyme is cut product after agarose gel electrophoresis, and glue reclaims object fragment and expression vector, reclaims product through T ₄dNA ligase(is purchased from NEB) connect.

Double digestion system:

10×Tango buffer 7.5ul

BamH I 3.75ul

EcoR I 3.75ul

Recombinant clone plasmid or expression vector 40.0ul

DH ₂o supplies volume to 75ul.

Endonuclease reaction system is acted on to 5h under 37 DEG C of conditions, then cut product with agarose gel electrophoresis qualification enzyme and rubber tapping is reclaimed, reclaim test kit (purchased from green skies reagent company) with DNA gel and reclaim product.

Linked system:

10×T4 ligase buffer 2ul

Recombinant cloning vector double digestion fragment 4ul

Expression vector double digestion fragment 12ul

T ₄DNA ligase 1ul

DH ₂o supplies volume to 25ul.

Linked system connects and spends the night at 16 DEG C, to connect product transfection Escherichia coli JM109 competent cell, coating is containing the LB flat board of penbritin, 37 DEG C of cultivations, choose respectively 10 single bacterium colonies, in the LB liquid nutrient medium that contains penbritin, cultivate, overnight incubation, extracting plasmid, PCR detects positive recombinant plasmid, and this enzyme is cut and is connected experiment and builds altogether 9 kinds of recombinant expression vectors and be respectively: PET32-IGF-1 is 1.; PET32-IGF-1 2.; PET32-IGF-1 people source; PET22-IGF-1 1.; PET22-IGF-1 2.; PET22-IGF-1 people source; PGEX4T-IGF-1 1.; PGEX4T-IGF-1 2.; PGEX4T-IGF-1 people source, wherein PET22 serial carrier is secreting, expressing, PET32 and PGEX4T-1 are intracellular expression.

The restriction enzyme mapping of recombinant clone plasmid PUC57-IGF-1 and expression plasmid carries out agarose gel electrophoresis detected result and sees Fig. 2.In Fig. 2, M:DL2000DNA molecular weight standard; 1:PET22b plasmid control group; 2:BamHI and EcoRI double digestion PET22b; 3:PET32a plasmid control group; 4:BamHI and EcoRI double digestion PET32; 5:PUC57-IGF-1 is plasmid control group 1.; 6:BamHI and 1. plasmid of EcoRI double digestion PUC57-IGF-1; 7:PUC57-IGF-1 is plasmid control group 2.; 8:BamHI and EcoRI double digestion PUC57-IGF-1 are 2.; The PGEX4T-1 plasmid of 9:PGEX4T-1 plasmid control group: 10:BamHI and EcoRI double digestion; The former plasmid control group in 11:PUC57-IGF-1 people source; The PUC57-IGF-1 people source of 12:BamHI and EcoRI double digestion.

Experimental result: as shown in Figure 2, carrier and goal gene success are by BamHI and EcoRI double digestion, and the IGF-1 gene stripe size that enzyme is cut is 243bp.

PCR reaction solution system:

PGEX4T-IGF-1 recombinant plasmid 4ul

Primers F 1ul

Primer R 1ul

Taq enzyme 12ul

DH ₂o supplies volume to 25ul.

PCR reaction system:

94 DEG C of 4min of denaturation

94 DEG C of 30s of sex change

55 DEG C of 30s return goods

Extend 72 DEG C of 3min

Be incubated 72 DEG C of 10min

Preserve 16 DEG C of 5min

Wherein, after sex change, the return of goods, the circulation of extension process 35 times, enter insulating process, PCR product identifies through agarose gel electrophoresis, and electrophoresis result is shown in Fig. 3.In Fig. 3, M:DL2000DNA molecular weight standard; 1:PET22-IGF-1 is recombinant plasmid PCR 1.; 2:PET22-IGF-1 is recombinant plasmid PCR 2.; The former restructuring plasmid PCR in 3:PET22-IGF-1 people source; 4:PET32-IGF-1 is recombinant plasmid PCR 1.; 5:PET32-IGF-1 is recombinant plasmid PCR 2.; The former restructuring plasmid PCR in 6:PET32-IGF-1 people source; 7:PGEX4T-IGF-1 is recombinant plasmid PCR 1.; 8:PGEX4T-IGF-1 is recombinant plasmid PCR 2.; 9: blank group of (dH ₂o replaces plasmid) PCR; The former restructuring plasmid PCR in 10:PGEX4T-IGF-1 people source.

Screening positive clone, cultivates each test strain, and extracting recombinant plasmid dna, after Auele Specific Primer PCR, is sent to the order-checking of Shanghai biotechnology company limited, and sequencing result feedback is constructed that gene order is entirely true.

3, the structure of recombinant expressed bacterium

By constructed 9 kinds of recombinant plasmids and empty plasmid Transformed E scherichia coli BL21 (DE3), Rosetta (DE3) and Origami (DE3) respectively, the common 36 strain test organismss that to obtain.

The abduction delivering of recombinant protein

(1) single bacterium colony of the 27 strain bacterium that picking is recombinated respectively, is inoculated in overnight incubation in the LB liquid nutrient medium that contains penbritin (100ug/mL) and paraxin (50ug/mL), and 37 DEG C, 180r/min, overnight incubation;

(2) get above-mentioned cultured bacterium liquid and be inoculated in the LB liquid nutrient medium that liquid amount is 10ml/50ml (containing penbritin (100ug/mL) and paraxin (50ug/mL)) by 8%, 37 DEG C, 180r/min, is cultured to OD ₆₀₀reach 0.5～0.6;

(3) be the IPTG abduction delivering of 0.1mmol/L to adding final concentration in step (2) gained bacterium liquid, 34 DEG C of inducing temperatures, induction time 8h, obtains fermented liquid, control group is separately set for not adding IPTG induction;

(4) with pipettor draw fermented liquid that step (3) obtains in 1.5ml centrifuge tube under 4 DEG C, 12000r/min condition centrifugal 3min, abandoning supernatant, repeats twice;

(5) with PBS damping fluid (0.05M, pH7.2) washing, centrifugal 3min under 4 DEG C, 12000r/min condition, abandoning supernatant, repeats twice;

(6) in sample, add 2 × buffer40ul, heating 15min;

(7) expression level of recombinant protein in the sample obtaining in employing SDS-PAGE electrophoretic analysis step (6);

(8) after recombinant expression plasmid conversion expression, carry out detected result through SDS-PAGE electrophoresis and see Fig. 4.

In Fig. 4, M: molecular weight of albumen standard; 1:PET32-IGF-1 is Transformed E scherichia coli BL21 (DE3) bacterial strain 1.; 2:PET32-IGF-1 is Transformed E scherichia coli BL21 (DE3) bacterial strain 2.; 1. 3:PET22-IGF-1 transforms Origami (DE3) bacterial strain; 1. 4:PGEX4T-IGF-1 transforms Rosetta (DE3) bacterial strain; 2. 5:PGEX4T-IGF-1 transforms Rosetta (DE3) bacterial strain; 6:PGEX4T-1 transforms Rosetta (DE3) bacterial strain; 7:PET32-IGF-1 people source former Transformed E scherichia coli BL21 (DE3) bacterial strain; The former conversion in 8:PET22-IGF-1 people source Origami (DE3) bacterial strain; The former conversion in 9:PGEX4T-IGF-1 people source Rosetta (DE3) bacterial strain.

Experimental result shows:

A. the 27 strain engineering bacterias that build, containing the former sequence in IGF-1 people source, 1. sequence and 2. each 9 strains of bacterial strain of sequence of IGF-1 of IGF-1, wherein have 19 strains can express target protein, and target protein is not expressed in all the other 8 strains;

B. there are 4 strains can express target protein containing in the intestinal bacteria of IGF-1 people source protogene, have 5 strains not express target protein, the bacterial strain that wherein expression amount is the highest is containing Rosetta (DE3) bacterial strain of the former expression plasmid in PGEX4T-IGF-1 people source and to name this bacterial strain be RPY source;

C. 1. in the intestinal bacteria of gene, there are 8 strains can express target protein containing preference IGF-1, there is 1 strain not express target protein, the bacterial strain that wherein expression amount is the highest is that the intestinal bacteria of not expressing goal gene are to contain 1. Escherichia coli BL21 (DE3) bacterial strain of expression plasmid of PGEX4T-IGF-1 containing 1. Escherichia coli BL21 (DE3) bacterial strain of expression plasmid of PET32-IGF-1;

D. 2. in the intestinal bacteria of gene, there are 7 strains can express target protein containing preference IGF-1, there are 2 strains not express target protein, the bacterial strain that wherein expression amount is the highest is containing the 2. Rosetta of expression plasmid (DE3) bacterial strain of PGEX4T-IGF-1, the intestinal bacteria of not expressing goal gene be contain PGEX4T-IGF-1 2. expression plasmid Escherichia coli BL21 (DE3) bacterial strain and contain the 2. Rosetta of expression plasmid (DE3) bacterial strain of PET22-IGF-1;

E. from the conclusion of a-d, the different I GF-1 codon of expressing same amino acid in same bacterial strain and same plasmid expression amount and whether can express different, wherein 1. gene the most easily expression in test host of preference IGF-1; 2. preference IGF-1 takes second place; Containing the most difficult expression in the Host Strains of experiment of IGF-1 people source protogene, prove that IGF-1 eukaryotic gene is subject to the Preference of codon to affect larger in prokaryotic hosts is expressed.

F. during taking PGEX4T-1 as the former sequence in vector expression IGF-1 people source, in Escherichia coli BL21 (DE3) bacterial strain, do not express goal gene; 1. in Escherichia coli BL21 (DE3) bacterial strain, do not express goal gene when sequence taking PGEX4T-1 as vector expression preference IGF-1; 2. in Escherichia coli BL21 (DE3) bacterial strain, do not express goal gene when sequence taking PGEX4T-1 as vector expression preference IGF-1;

G. during taking PET22b as the former sequence in vector expression IGF-1 people source, in Rosetta (DE3), Origami (DE3) and Escherichia coli BL21 (DE3) bacterial strain, do not express goal gene; 1. in all test strains, all can express goal gene when sequence taking PET22b as vector expression preference IGF-1; 2. in Rosetta (DE3) bacterial strain, do not express goal gene when sequence taking PET22b as vector expression preference IGF-1;

H. during taking PET32a as the former sequence in vector expression IGF-1 people source, in Escherichia coli BL21 (DE3) bacterial strain, do not express goal gene; 1. in all test strains, all can express goal gene when sequence taking PET32a as vector expression preference IGF-1; 2. in all bacterial strains, all can reach goal gene when sequence taking PET32a as vector expression preference IGF-1;

I. from the conclusion of f-h, whether same expression plasmid is expressed the expression amount of different I GF-1 codon and can be expressed differently in bacterial strain of the same race, wherein in test host, the most easily expresses target protein using PET32a as vector expression IGF-1; PGEX4T-1 takes second place at test Host Strains as vector expression IGF-1; PET22b the most difficult expression in the Host Strains of test, evidence the ability to express difference of same plasmid IGF-1 sequence that different codons are formed, wherein in Rosetta (DE3) bacterial strain, to express target protein ability the strongest for PGEX4T-1 and the IGF-1 recombinant plasmid that 2. gene forms, and name this bacterial strain be RPP 2..

J., while expressing the former sequence in IGF-1 people source with Escherichia coli BL21 (DE3) bacterial strain, this bacterial strain can not be expressed containing the PGEX4T-I plasmid of the former sequence in IGF-1 people source and PET32a-IGF-1 plasmid; Express IGF-1 1. when sequence with Escherichia coli BL21 (DE3) bacterial strain, this bacterial strain can not be expressed the 1. PGEX4T-1 plasmid of sequence containing IGF-1; Express IGF-1 2. when sequence with Escherichia coli BL21 (DE3) bacterial strain, this bacterial strain can not be expressed the 2. PGEX4T-1 of sequence containing IGF-1;

K., while expressing the former sequence in IGF-1 people source with Rosetta (DE3) bacterial strain, this bacterial strain can not be expressed the PET22b plasmid containing the former sequence in IGF-1 people source; Express IGF-1 1. when sequence with Rosetta (DE3) bacterial strain, this bacterial strain can be expressed and be contained 1. all PGEX4T-1 plasmids of sequence of IGF-1; Express IGF-1 2. when sequence with Rosetta (DE3) bacterial strain, this bacterial strain can not be expressed the 2. PET22b plasmid of sequence containing IGF-1;

M., while expressing the former sequence in IGF-1 people source with Origami (DE3) bacterial strain, this bacterial strain can not be expressed the PET22b plasmid containing the former sequence in IGF-1 people source; Express IGF-1 1. when sequence with Origami (DE3) bacterial strain, this bacterial strain can be expressed and be contained 1. all plasmids of sequence of IGF-1; Express IGF-1 2. when sequence with Origami (DE3) bacterial strain, this bacterial strain can be expressed and be contained 2. all plasmids of sequence of IGF-1;

N. from the conclusion of j-m, whether same expression strain is expressed the expression amount of different I GF-1 codon and can be expressed differently in plasmid of the same race, and wherein Origami (DE3) bacterial strain is the most easily expressed IGF-1 gene order, but expression amount is generally not high; Rosetta (DE3) bacterial strain amount is the highest, wherein the high expression level amount of the former sequence in IGF-1 people source and preference IGF-1 sequence is all expressed by this bacterial strain, Escherichia coli BL21 (DE3) bacterial strain is least applicable to the expression of IGF-1 gene, the kind that not only can express is minimum, and expression amount is also very low.

O. a-n draws different IGF-1 codon sequences; Expression plasmid and expression strain all have considerable influence to the expression of target protein, from this experiment, filter out can express preference IGF-1 sequence high expression level bacterial strain RPP 2., the high expression level bacterial strain RPY source of expressing people source former IGF-1 sequence, 2. experiment will be expressed target protein with RPY source as the bacterium that sets out using bacterial strain RPP and detect for HPLC.

4, the expression amount of Liquid Detection target protein

(1) containing the 2. Rosetta of recombinant vectors (DE3) bacterial strain and 37 DEG C of cultivation 6h of Rosetta (DE3) bacterial strain containing PGEX4T-IGF-1 people source recombinant vectors of PGEX4T-IGF-1, use final concentration 0.5mMIPTG, 32 DEG C of abduction delivering 8h;

(2) sample centrifugal, bacteriolyze, homogeneous, centrifuging and taking supernatant NH after broken wall ₄sO ₄the centrifugal supernatant of abandoning of 10000rpm after saltouing;

(3) precipitation that step (2) obtains mixes with balance liquid by a certain percentage, after dissolving completely, loading is crossed NI post, with after imidazoles wash-out, collecting elutriant adds enteropeptidase to be placed in dialysis tubing enzyme by 1:750 to cut and spend the night, NI column purification, sample is crossed the degerming of 0.22um sterile film, and HPLC detects, and detects collection of illustrative plates and sees Fig. 5～Fig. 7.

Experimental result is in table 2:

The each sample detection result of table 2

Sample title	Extension rate	Peak area	Standard specimen area	Concentration
					Standard model	×		24474401	50ug/mL
IGF-1 people source	2	13642337	24474401	55.7ug/mL
					IGF-1②	2	32272187	24474401	131.89ug/mL

Analyze: from the data of Fig. 5～Fig. 7 and table 2, Rosetta (DE3) bacterial strain of constructed PGEX4T-IGF-1 people source recombinant vectors can be expressed the IGF-1 albumen of 55.7ug/mL, under the same conditions containing PGEX4T-IGF-1 2. the Rosetta of recombinant vectors (DE3) bacterial strain can express the IGF-1 albumen of 131.89ug/mL, 2.4 times of human source gene sequence expression amount, the IGF-1 bacterial strain that has successfully built high expression level is invented in this research, if this research successfully can directly be obtained to the profit of nearly 2.4 times on the original basis for suitability for industrialized production, and this research is invented also for the improvement of other industrial strains provides experiment and data basis.

Claims

1. one kind builds the engineering bacteria of high expression level IGF-1 gene based on protokaryon codon preference, it is characterized in that: described engineering bacteria is can be at the genetic engineering bacterium of intracellular expression or excreting and expressing recombinant human insulin-like growth factor fusion proteins, described engineering bacteria is colibacillus engineering, and the aminoacid sequence of described fusion rotein is shown in Seq ID No:4.

2. the engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference according to claim 1, is characterized in that: described engineering bacteria comprises the recombinant expression vector and the expression strain that contain IGF-1 gene.

3. the engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference according to claim 2, it is characterized in that: the carrier that sets out of described recombinant expression vector is that its gene order is shown in Seq ID No:1 without the IGF-1 people source protogene sequence of optimizing.

4. the engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference according to claim 1, it is characterized in that: 1. described IGF-1 gene comprises complete synthesis IGF-1 Preference order after codon optimized, and its gene order is shown in Seq ID No:2.

5. the engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference according to claim 1, it is characterized in that: 2. described IGF-1 gene comprises complete synthesis IGF-1 Preference order after codon optimized, and its gene order is shown in Seq ID No:3.

6. the engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference according to claim 2, is characterized in that: described recombinant expression vector comprises intracellular expression carrier and secretion expression carrier.

7. the engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference according to claim 6, it is characterized in that: described intracellular expression carrier be by IGF-1 Preference order 1. or IGF-1 Preference order 2. DNA molecular be inserted into the recombinant plasmid that the multiple clone site place of plasmid PGEX4T-1 and PET32a obtains, in described intracellular expression carrier, starting the promotor that described DNA transcribes is T7 promotor.

8. the engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference according to claim 6, it is characterized in that: described secretion expression carrier be by IGF-1 Preference order 1. or IGF-1 Preference order 2. DNA molecular be inserted into the recombinant plasmid that the multiple clone site place of plasmid PET22b obtains, in described secretion expression carrier, starting the promotor that described DNA transcribes is T7 promotor.

9. the engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference claimed in claim 1, is characterized in that: described intestinal bacteria comprise Escherichia coli BL21 (DE3), Rosetta (DE3) and Origami (DE3).

10. the engineering bacteria that builds high expression level IGF-1 gene based on protokaryon codon preference claimed in claim 2, it is characterized in that: described expression strain comprises the engineering strain of a strain through Preference transformation, 2., the RPP 2. expression amount of target protein is constructed 2.4 times without the high expression level amount of transformation people source protogene engineering bacteria to this project bacterial strain called after RPP.