CN101724641A - Method for preparing human serum amyloid A1 and expression vector and genetic engineering bacteria thereof - Google Patents

Abstract

The invention provides a method for preparing soluble genetic engineering human serum amyloid A1 (SAA1) and an expression vector and genetic engineering bacteria thereof. The expression vector provided by the invention is a plamid vector pET28a(+) containing a human SAA (hSAA1) gene sequence, and the hSAA1 encoding sequence is positioned between restriction enzyme cutting sites of NdeI and Xhol I on the pET28a(+) vector. The genetic engineering bacteria provided by the invention are colibacillus pET28a (+)-hSAA1/Rosetta-gami, which can express soluble target protein. The preparation method of the invention uses a nickel cross-linking affinity-column one-step method, has simple operation, high yield and short consumed time, and is suitable for industrial large-scale production, and the purity of the obtained target protein is larger than 90%.

Description

The method and expression vector and the genetic engineering bacterium that prepare human serum amyloid A 1

Technical field

The present invention relates to utilize the DNA recombinant technology to produce the field of protein or polypeptide, more specifically, the present invention relates to genetically engineered and prepare human serum amyloid A 1 (Serum Amyloid A1, method SAA1) also relates to its expression vector and genetic engineering bacterium.

Background technology

(Serum Amyloid A SAA) is a kind of acute phase reaction lipophorin to serum amyloid A protein, and the SAA gene is positioned on No. 11 karyomit(e), is one group of polymorphism albumen by same cluster gene coding.Human serum amyloid A 1 (SAA1) is made up of 104 amino acid, about the about 12KD-14KD of molecular weight.SAA is a polymorphism albumen, is made up of several relevant protein families (SAA1-SAA4).Mainly synthetic by liver cell.Wherein the gene of SAA1 and SAA2 has only 7 amino acid whose difference, their the acute phase SAA albumen of all encoding.SAA4 changes in acute phase reaction not quite, is a special-shaped thing that occurs with high density lipoprotein cholesterol in running balance.

SAA and C-reactive protein (CRP) all are acute phase reactive proteins, and all can be subjected to cytokine stimulates and express, but SAA is more responsive to stimulating.Early stage in acute infection, SAA is subjected to inflammatory factors such as IL-1, IL-6 and TNF to be stimulated, synthetic in a large number in liver cell, the specificity land of its N end and high-density lipoprotein (HDL) (HDL) form the SAA-HDL particle then, and then the metabolism of adjusting high-density lipoprotein (HDL), to strengthen with scavenger cell avidity and to carry lipid to inflammation part, SAA is effective neutrophil activation agent, and the anti-microbial effect, the mould fungus inhibition that strengthen neutrophil leucocyte infect.In addition, SAA also shows the similar characteristics of cytokine, can bring out ILlb and produce.Equally, SAA also raises T lymphocytic emiocytosis Interferon, rabbit.SAA can reduce the generation of metalloprotease, thus transmitting tissue's injury repairing.

More meaningfully, being different from CRP only changes when acute inflammation, SAA all changes at acute and chronic inflammation patients serum intensive amount, sometimes can reach 1000 times (normal value is generally 910 ± 270 micrograms per litre) of normal concentration, therefore, SAA is more responsive as the biological detection index of reflection infectious diseases inflammation.Clinical meaning and application for SAA receive very big concern, find that as the researchist of Univ Melbourne Australia SAA is not only the biological markers of chronic obstructive pulmonary disease (COPD) acute exacerbation phase, also be the predictor of acute myocardial infarction (AMI) patient generation cardiac rupture after accepting the emergency treatment coronary artery to get involved (PCI) operation, this provides the important references index for early discovery cardiac rupture patient.Other a large amount of researchs show that also the SAA rising has very confidential relation with disease activity in pancreatitis, hepatitis, coronary heart disease, diabetes, chronic renal disease, rheumatoid arthritis and fat case.Therefore, clinical study is at present thought, compare with other acute phase reactive protein, SAA is the sensitiveest in the acute inflammation stage reaction, the albumen of appreciation amplitude maximum, particularly be suppressed and go down and chronic inflammatory diseases when causing the situation that CRP level in the body can not reflect that disease changes in immunologic function, SAA still keeps good dependency with the generation development of inflammatory disease, and the CRP and the SAA level that detect the patient simultaneously can improve diagnosis of infection sensitivity.And SAA is more accurate than CRP in diagnosis of viral infection, kidney transplantation exclusion reaction and aspect the healing of adrenocortical hormones in treating cystic fibrosis.Research simultaneously shows that also the tumor correlated albumen of SAA and alpha-fetoprotein etc. is similar, its concentration and positive correlation of cancer generation journey in serum.Therefore the detection of content in the patients serum has auxiliary diagnosis value to SAA clinically.

SAA is not a kind of simple marker of inflammation, actively gets involved the correlated process of disease yet.Degraded product such as SAA is made up of 76 amino acid, and molecular weight is 8500 amyloid A proteins (AA), and it is deposited between each histoorgan usually, is mainly seen in chronic infection, the general amyloid pathology that inflammation or tumour cause.In glaucomatous research, find equally, because SAA causes that partial eyeball pressure is excessive, thereby brings out the glaucoma disease at the overexpression of eye.Owing to SAA relevant this characteristic of generation with multiple disease, the methods of treatment at SAA more and more comes into one's own at present.

In view of the importance of SAA in clinical treatment and diagnosis, both at home and abroad the research of SAA is more and more paid attention at present.Generally use two kinds of expression systems at present: intestinal bacteria and insect expression system, express GST-SAA and rSAA albumen respectively.But the in-vitro recombination expression of SAA and purifying are owing to its protein characteristic, and as its easy aggregation in vitro, expression amount is low, and the soluble proteins of acquisition is few, purification process complexity etc.Thereby limited further carrying out of SAA in vitro study for a long time always.The reorganization SAA albumen that obtains by these two kinds of methods is inclusion body, therefore in purge process, must use 6M urea or Guanidinium hydrochloride that the purifying target protein is become complicated means such as renaturation, thereby the recombinant protein biological activity that purifying obtains is lower, and active unstable.In addition, this purification process length consuming time, yield is low, cost is high, is unfavorable for mass production.

Disclosed preparation method there is no report at home and abroad, the present invention is by the method for gene amplification and reorganization, mature peptide at SAA1 is cloned reorganization, and add poly Histidine (6x His) affinity peptide mark at N-end, and then select for use intestinal bacteria Rosetta-gami as the host bacterium, obtained soluble target protein.The invention has the advantages that (1) is the solubility maturation protein in order to the reorganization SAA albumen that last method obtains, thereby has avoided the change renaturation in purification step, reduction its pleated sheet structure in vivo in maximum likelihood ground keeps its biological activity; (2) can simplify purification process to the use of recombinant mature SAA-poly Histidine affinity peptide; (3) use intestinal bacteria Rosetta-gami, the SAA albumen great expression that prevents to greatest extent to recombinate is to the toxicity of host cell, thereby improves the output of recombinant protein.

Summary of the invention

The present invention provides a kind of at expression in escherichia coli soluble human SAA1, i.e. human serum amyloid A 1, and can be by the recombination method of this soluble human of affinity column efficient production SAA1, and expression vector and engineering bacteria.

A first aspect of the present invention, the expression vector of a kind of genetically engineered people SAA1 is provided, this carrier contains the plasmid vector pET28a (+) of people SAA (hSAA1) gene order, and the hSAA1 encoding sequence is positioned on pET28a (+) carrier between the NdeI and XholI restriction enzyme site.

A second aspect of the present invention provides a kind of genetic engineering bacterium, is pET28a (+)-hSAA1/Rosetta-gami, and its host is intestinal bacteria (Escherichia coli) Rosetta-gami, and expression vector described by the present invention transforms.

A third aspect of the present invention provides a kind of and expresses and the preparation method of purifying gene engineering people SAA, comprises the steps: 1. to cultivate and expressing gene engineering bacteria pET28a (+)-hSAA1/Rosetta-gami; 2. centrifugal collection genetic engineering bacterium; 3. the affinity column purifying of fusion rotein SAA1; 4. the desalination of dialysing obtains target protein.

Preparation method of the present invention is the host bacterium with intestinal bacteria (Escherichia coli) Rosetta-gami, described carrier is selected the plasmid vector pET28a (+) that contains people SAA (hSAA1) gene order for use, and the hSAA1 encoding sequence is positioned on pET28a (+) carrier between the NdeI and XholI restriction enzyme site.By gene amplification and reorganization, clone reorganization at the mature peptide of SAA1, remove 20 amino acid of the N end of SAA precursor protein, and add 6 affine polypeptide markers of poly Histidine at its N end, constitute the SAA-6His fusion rotein.By nickel affinity column single step purification, obtain the target protein of solubility.

Nickel affinity column single step purification of the present invention, purification step is simple, and required time is short, and the omnidistance time of purifying is shorter, and the omnidistance time of preferred purifying is 4-6 hour.The long-pending relation of the bacteria liquid of omnidistance time of purifying optimization and purifying is generally: the expression bacterium liquid of 1 liter (1L), the purifying time needs about 4 hours.

" SAA precursor protein " of the present invention is meant the coding by SAA cDNA, synthetic in cell, the SAA full-length proteins of not excising signal peptide." mature peptide of SAA1 " of the present invention is meant and excises signal peptide in cell, is secreted in the serum, has the SAA albumen of biological function.

" nickel affinity column single step purification " of the present invention is meant only by a nickel affinity column, by appropriate method, directly from the needed target protein of affinity column acquisition.Not needing to rely on additive method does not need to rely on other reagent and is further purified.

Innovation part of the present invention is: adopt the people SAA1 maturation protein after poly Histidine fusion expression vector (PET28a) is expressed the removal signal peptide first, and the one, can be at a large amount of soluble people SAA1 albumen of e. coli expression.The solubility target protein that is obtained does not need to become additive methods such as renaturation step, purification process is oversimplified, and the yield of target protein greatly improves.The 2nd, 6 poly Histidine polypeptide of N end that can utilize fusion rotein are as the purifying mark, and are fast and convenient efficient, help follow-up purifying process, obtain the highly purified people SAA1 albumen of recombinating, and its purity is more than 95%.Purifying cost of the present invention is low, but the purity height can obtain the solubility reorganization SAA albumen more than the 10mg in 1 liter of engineering bacteria liquid.

SAA purification process more of the present invention and traditional SAA purification process, the result as shown in Table 1.

Table one: the comparison of SAA purification process of the present invention and traditional SAA purification process

The invention has the advantages that (1) is the solubility maturation protein in order to the reorganization SAA albumen that last method obtains, thereby has avoided the change renaturation in purification step, reduction its pleated sheet structure in vivo in maximum likelihood ground keeps its biological activity; (2) can simplify purification process to the use of recombinant mature SAA-poly Histidine affinity peptide; (3) use intestinal bacteria Rosetta-gami, the SAA albumen great expression that prevents to greatest extent to recombinate is to the toxicity of host cell, thereby improves the output of recombinant protein.

In sum, use recombinant vectors of the present invention, express engineering bacteria and method of purifying protein, can easyly obtain purity apace and reach soluble human vitro recombination SAA1 protein more than 95%.

Description of drawings

Fig. 1: reverse transcription PCR amplification in the tissue of patient obtains people SAA1.Wherein, M: standard DNA ladder; 1-3: the PCR product of differential responses pipe.

The enzyme of Fig. 2: pET28a (+)-hSAA1 recombinant expression plasmid is cut evaluation.Wherein, M: standard DNA ladder; 1-14: the different cloned plasmids (restriction enzyme is NedI, XholI) that double digestion is identified.

Fig. 3: total length PET28a-rSAA recombinant plasmid collection of illustrative plates.

Fig. 4 a: recombinant human SAA albumen is IPTG abduction delivering electrophorogram (15% acrylamide gel) in Rosetta-gami host bacterium.Wherein, M: standard protein ladde, before 1:IPTG induces; 2-4: different SAA-rosetta-gami host bacterium IPTG induced back 2.5 hours.

Fig. 4 b: before each SAA expressive host bacterium IPTG induces after induce, host's bacteria growing concentration OD value graphic representation.The result shows from bacterium liquid growth curve, and SAA-Rosetta-gami IPTG induces back growth best, and the Rosetta-gami ratio is more suitable for the people SAA that recombinates and expresses.

Fig. 5: 15% SDS-PAGE detects the final recombinant protein that obtains.Total bacterium lysate before wherein, lane1:IPTG induces; Total bacterium lysate after lane2:IPTG induces; Lane3: the product behind the ni-sepharose purification.

Fig. 6: Western blot (Weston Blot) test.Total bacterium lysate before wherein, lane1:IPTG induces; Total bacterium lysate after lane2:IPTG induces; Lane3: the product behind the ni-sepharose purification.

Fig. 7: purifying protein is gone up the result of purity check at high performance liquid chromatography (HPLC).Analyzing the purity of protein that records through HPLC is: 97.25%.

Fig. 8: albumen molecular weight analyse result on the mass spectrum of purifying protein.Reorganization SAA1 protein molecular weight is determined as through mass spectrometer: 13.8KD.

Fig. 9: mass spectrum is to the amino acid polypeptide analytical results of purifying protein.Show that by amino acid polypeptide analysis and reduction result this recombinant protein sequence is consistent with target protein (people SAA1 mature peptide) sequence, purity 97.27% to this recombinant protein of purifying.

Specific implementation method

Below in conjunction with embodiment and accompanying drawing, further set forth the present invention.Below each embodiment be for the explanation the present invention but not for the restriction scope of the invention.

The encoding gene pcr amplification of embodiment 1. people SAA1 and the structure of recombinant expression plasmid pET28a (+)-SAA

Construction step is as follows:

1) extracted total RNA:

Get fresh patient's liver organization, homogenizer is pulverized back RNA extraction agent box extracted total RNA.

2) pcr amplification of goal gene (RT-PCR):

With total RNA is masterplate, uses the reverse transcription test kit, obtains the cDNA of SAA1.According to a pair of primer of announcing in the gene pool of people SAA1 sequences Design, primer is:

Upstream primer: 5 ' AT CATATG TTCTTTTCGTTCCTTGGCGAG (Nde I);

Downstream primer: 5 ' AT CTCGAG TCAGTATTTCTCAGGCAGG (Xhol I).

With reference to the people SAA1 sequence of having reported, from the 60th Nucleotide of cDNA, the proteic mature peptide gene of people SAA increases during the design primer.

Adopt following PCR reactive system and program: in a 200ul trace P CR reaction tubes, add following reagent: 10XPCR damping fluid: 5 μ l; Masterplate cDNA storehouse: 1 μ g; DNTP:1mM; Each 100pM of upstream and downstream primer; Taq enzyme: 2U; Add the deionization aqua sterilisa to final volume 50ul.Reaction conditions be 94 ℃ 5 minutes, 94 ℃ 30 seconds, 48 ℃ 30 seconds, 72 ℃ 30 seconds, 40 circulations, 72 ℃ were extended 10 minutes then.

After reaction is finished, reaction product with 1.5% sepharose TAE electrophoresis, is seen accompanying drawing 1, the DNA cloning fragment that the result obtains is consistent with the gene size of desired design.

3) PCR product cloning:

The PCR fragment that amplifies, reclaiming test kit with the PCR product reclaims, double digestion then, agarose gel electrophoresis, be connected on pET-28a (+) carrier with direct orientation behind the glue recovery test kit purifying, method is seen document (Sambrooks, the molecular cloning handbook), to connect product is transformed in the DH5a host bacterium, extract plasmid, identify agarose electrophoresis result with NdeI and XholI double digestion, as shown in Figure 2, endonuclease bamhi size (fragment that be about 300 Nucleotide) positive clone consistent with design.

4) positive colony is carried out determined dna sequence:

The positive colony that screening is obtained carries out DNA extracting on a small scale, and method is seen document (Sambrooks, molecular cloning handbook), and extractive DNA plasmid is delivered to order-checking company check order, and as sequence 1, shown in the SEQ NO.1, sequencing primer is the T7 universal primer.Total length PET28a-rSAA recombinant plasmid collection of illustrative plates is seen shown in the accompanying drawing 3.Total length PET28a-rSAA recombinant plasmid nucleotide sequence, as sequence 2, shown in the SEQ NO.2, wherein, the black matrix line part among the SEQNO.2 is for inserting people saa1 gene order.Its sequencing result and its gene pool have been reported the comparison (NM 000331) of people SAA1 sequence, and sequence is relatively more consistent, as sequence 3, shown in the SEQ NO.3.

The structure and the screening of embodiment 2. efficient expression engineerings

Recombinant plasmid pET-28 (a)-SAA is transformed among the intestinal bacteria Rosetta-gami, gets the single colony inoculation of reorganization bacterium and contain in the LB substratum of kantlex 37 ℃ of shaking table overnight incubation in 3ml.Next day, the recombinant bacterial strain with incubated overnight was inoculated in the 3mlLB substratum by 2%, and 37 ℃ of shaking tables are cultivated about 2 hours when OD600 is 0.8 left and right sides, add IPTG to final concentration 1mM, induce 2.5 hours.15% SDS-PAGE detects Expression of Fusion Protein form and expression amount, the screening efficient expression strain.The results are shown in the following Table two, accompanying drawing 4a and accompanying drawing 4b.Accompanying drawing 4a and 4b show, show from protein electrophoresis figure and host's bacteria growing curve result, adding the total incubation time of inductor IPTG after 1.5 hours, the growth of other host bacterium all shows inhibition even stops, host bacterium Rosetta-gami is upgrowth situation the best then, the most suitable express recombinant people SAA albumen.

Table two: each one SAA-expresses bacterial classification, and different time growth concentration OD600 measures

The control group time	??BL21D??E3/LB	??rosetta-gami??/LB	??plysS/??LB	??BL21DE3/??2YT	??rosetta-gami/??2YT	??plysS/2??YT
							??0min	??0	??0	??0	??0	??0	??0
??20min	??0.104	??0.079	??0.09	??0.098	??0.084	??0.098
							??40min	??0.294	??0.219	??0.254	??0.248	??0.22	??0.247
??1hour	??0.503	??0.4	??0.475	??0.495	??0.423	??0.479
							??1.5hour	??0.755	??0.602	??0.671	??0.763	??0.69	??0.741
??2hour	??0.876	??0.785	??0.813	??0.943	??0.935	??0.875
							??2.5hour	??0.97	??0.954	??0.842	??0.748	??1.183	??0.708

The control group time	??BL21D??E3/LB	??rosetta-gami??/LB	??plysS/??LB	??BL21DE3/??2YT	??rosetta-gami/??2YT	??plysS/2??YT
							??3hour	??0.878	??0.959	??0.685	??0.472	??1.241	??0.397
??3.5hour	??0.791	??0.94	??0.376	??0.242	??1.272	??0.209
							??4hour	??0.551	??0.953	??0.076	??-0.004	??1.224	??0.047
??4.5hour	??0.258	??0.887	??-0.145	??-0.2	??1.137	??-0.144

Annotate: when the OD value reaches 0.8, take out bacterium liquid adding 1mM IPTG and induce and continued growth.

The extensive expression and the purification process of embodiment 3. gene recombination bacteriums

The expression of recombinant human SAA-poly Histidine fusion rotein in escherichia coli host

1) PET28a-saa1 is transformed in the Rosetta-gami intestinal bacteria on the LB culture plate that has kalamycin resistance (50ng/ml) 37 ℃ of overnight incubation;

2) single bacterium colony of picking is in the LB that 50ml contains 50 mcg/ml kantlex cultivates 37 ℃ 250 rev/mins, overnight incubation;

3) 2% inoculation culture (comprises the 50ug/ml kantlex) in 1L LB substratum, 37 ℃ of to A600, O.D.0.5;

4) add IPTG to 37 ℃ of final concentration 1mM, 300 rev/mins of inducing culture 3hours;

5) high speed centrifugation 4000rpm is 20 minutes, and collecting precipitation also is kept at-80 ℃.

The reagent of purification of recombinant human SAA-poly Histidine fusion rotein

1) lysate: 50mM Tris-HCl Ph 8.0,500mM NaCl, 10mM imidazole, 1mMPMSF, 5mM 2-Me

2) washings 1:50mM Tris-HCl Ph 8.0,500mM NaCl, 10mM imidazole, 1mMPMSF, 5mM 2-Me

3) washings r2:50mM Tris-HCl Ph 8.0,500mM NaCl, 20mM imidazole, 1mMPMSF, 5mM 2-Me

4) washings 3:50mM Tris-HCl Ph 8.0,500mM NaCl, 30mM imidazole, 1mMPMSF, 5mM 2-Me

5) elutriant 1:50mM Tris-HCl Ph 8.0,150mM NaCl, 100mM imidazole, 1mMPMSF, 5mM 2-Me

6) elutriant 2::50mM Tris-HCl Ph 8.0,150mM NaCl, 300mM imidazole, 1mMPMSF, 5mM 2-Me

Purification step

1) intestinal bacteria with 1 liter of LB substratum expression SAA1 recombination fusion protein are suspended in the 50ml lysate;

2) at ultrasonic 4-5 time on ice, 30 seconds/time;

3) the 10000g high speed centrifugation is 15 minutes;

4) get supernatant 10000g 10 minutes at a high speed again;

5) get the supernatant upper prop in 1ml nickel post;

6) clean the nickel post with 30ml washings 1;

7) clean the nickel post with 30ml washings 2;

8) clean the nickel post with 30ml washings 3;

9) with 3ml elutriant 1 wash-out target protein from the nickel post, collect a test tube for per 11;

10) with 3ml elutriant 2 wash-out target protein from the nickel post, collect a test tube for per 11;

11) with the sample mix of wash-out together, and use 1XPBS, 4 ℃ of dialysis are spent the night for three times;

12) will dialyse good sample retention at-80 ℃.

The expression of gene recombination bacterium and purification result, as shown in Figure 5, wherein, total bacterium lysate before lane1:IPTG induces; Total bacterium lysate after lane2:IPTG induces; Lane3: the product behind the ni-sepharose purification.By dialysis, the A280OD pH-value determination pH calculates: the albumen total concn is 2.4mg/ml; Be total to 6ml, net result is about 14mg albumen/L bacterium liquid expresses.

Embodiment 4. Western blottings are to the proof of recombinant protein

1) 15% the third rare acid amides glue, 90V, 3 hours

2) by the Tris-glycine damping fluid protein fragments electricity on third rare acid amides is forwarded on the pvdf membrane, condition is the 1Xtris-glycine damping fluid, 20% methyl alcohol, and under the 70V voltage, room temperature shifted 3 hours

3) pvdf membrane after the electricity commentaries on classics is taken out, cleaned 30 minutes with 1X PBST

4) with the PBST sealing that contains 5% skim-milk 45 minutes

5) use 1X PBST rinsing pvdf membrane 3 times, 10 minutes/time

6) adding contains the PBST 8mL of the anti-people SAA antibody of dilution in 1: 2000, room temperature reaction 1 hour

7) wash pvdf membrane three times with 1X PBST, 15 minutes/time

8) adding contains the antibody of the anti-mouse of dilution in 1: 1000, room temperature reaction 40 minutes

9) wash pvdf membrane three times with 1X PBST, 15 minutes/time

10) with containing 0.3%H ₂O ₂With the colour developing liquid of 6mg diaminobenzidine, the 15 seconds experimental results that develop the color as shown in Figure 6.

Embodiment 6. high performance liquid chromatography (HPLC) are to the purity check of purifying protein

The purifying protein that obtained among the above embodiment is gone up purity check at high performance liquid chromatography (HPLC), HPLC testing method routinely, its result as shown in Figure 7, the purity interpretation of result is 97.25% for detecting purity of protein.

Embodiment 7. mass spectrums are analyzed the molecular weight of albumen of purifying protein

The purifying protein that is obtained among the above embodiment is carried out molecular weight analyse on mass spectrum, mass spectrometric analysis method routinely, reorganization SAA1 protein molecular weight is determined as through mass spectrometer: 13.8KD, its result match with the molecular weight that calculates from aminoacid sequence as shown in Figure 8.

Embodiment 8. analyzes its amino acid polypeptide by mass spectrum

Show that by amino acid polypeptide analysis and reduction result as shown in Figure 9, this recombinant protein sequence is consistent with target protein (people SAA1 mature peptide) sequence, purity 97.27% to this recombinant protein of purifying.That is, use recombinant vectors of the present invention, express engineering bacteria and method of purifying protein, can obtain purity and reach human body more than the 95% SAA1 protein of recombinating outward.

Sequence table

＜110〉DESAY's diagnositc system (Shanghai) Co., Ltd.

＜120〉prepare method and the expression vector and the genetic engineering bacterium of human serum amyloid A 1

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＜213〉artificial sequence

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aggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgc

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cttcggtttccgtgtttcgtaaagtctggaaacgcggaagtcagcgccctgcaccattatgttccggatctgcatcgcaggat

gctgctggctaccctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttctctggtcccgcc

gcatccataccgccagttgtttaccctcacaacgttccagtaaccgggcatgttcatcatcagtaacccgtatcgtgagcatc

ctctctcgtttcatcggtatcattacccccatgaacagaaatcccccttacacggaggcatcagtgaccaaacaggaaaaaa

ccgcccttaacatggcccgctttatcagaagccagacattaacgcttctggagaaactcaacgagctggacgcggatgaac

aggcagacatctgtgaatcgcttcacgaccacgctgatgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtga

aaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcag

ggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggct

taactatgcggcatcagagcagattgtactgagagtgcaccatatatgcggtgtgaaataccgcacagatgcgtaaggaga

aaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcag

ctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagca

aaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatc

gacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgc

tctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgct

gtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcg

ccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggat

tagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtattt

ggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggta

gcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggt

ctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgaacaataaaactgtctgcttacataaacagtaatac

aaggggtgttatgagccatattcaacgggaaacgtcttgctctaggccgcgattaaattccaacatggatgctgatttatatgg

gtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagtt

gtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgc

ctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattcc

aggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgt

ttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagt

gattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccattctcaccggattca

gtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcgg

aatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaa

aaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaagaattaattcatgagcgga

tacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgaaattgtaaac

gttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataa

atcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaac

gtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgagg

tgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcg

agaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaacca

cccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccaacacc

<210>3

<211>309

<212>DNA

＜213〉artificial sequence

<400>3

Report sequence 133TTCTTTTCGTTCCTTGGCGAGGCTTTTGATGGGGCTCGGGACATGTGGAGAGC CTACTCT 192

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Extension increasing sequence 284TTCTTTTCGTTCCTTGGCGAGGCTTTTGATGGGGCTCGGGACATGTGGAGAGC CTACTCT 343

Report sequence 193GACATGAGAGAAGCCAATTACATCGGCTCAGACAAATACTTCCATGCTCGGGG GAACTAT 252

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Extension increasing sequence 344GACATGAGAGAAGCCAATTACATCGGCTCAGACAAATACTTCCATGCTCGGGG GAACTAT 403

Report sequence 253GATGCTGCCAAAAGGGGACCTGGGGGTGCCTGGGCTGCAGAAGCGATCAGCGA TGCCAGA 312

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Extension increasing sequence 404GATGCTGCCAAAAGGGGACCTGGGGGTGCCTGGGCTGCAGAAGCGATCAGCGA TGCCAGA 463

Report sequence 313GAGAATATCCAGAGATTCTTTGGCCATGGTGCGGAGGACTCGCTGGCTGATCA GGCTGCC 372

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Extension increasing sequence 464GAGAATATCCAGAGATTCTTTGGCCATGGTGCGGAGGACTCGCTGGCTGATCA GGCTGCC 523

Report sequence 373AATGAATGGGGCAGGAGTGGCAAAGACCCCAATCACTTCCGACCTGCTGGCCT GCCTGAG 432

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Extension increasing sequence 524AATGAATGGGGCAGGAGTGGCAAAGACCCCAATCACTTCCGACCTGCTGGCCT GCCTGAG 583

Report sequence 433AAATACTGA 441

|||||||||

Extension increasing sequence 584AAATACTGA 592

Claims (6)

1. the expression vector of a genetically engineered people SAA1 is characterized in that, this carrier contains the plasmid vector pET28a (+) of people SAA (hSAA1) gene order, and the hSAA1 encoding sequence is positioned on pET28a (+) carrier between the NdeI and Xhol I restriction enzyme site.

2. a genetic engineering bacterium is characterized in that, described genetic engineering bacterium is pET28a (+)-hSAA1/Rosetta-gami, and the host bacterium is intestinal bacteria (Escherichia coli) Rosetta-gami, is transformed by the described expression vector of claim 1.

3. a method for preparing human serum amyloid A 1 (hSAA1) is characterized in that, described method comprises the steps: 1. to cultivate and expressing gene engineering bacteria pET28a (+)-hSAA1/Rosetta-gami; 2. centrifugal collection genetic engineering bacterium; 3. the affinity column purifying of fusion rotein SAA1; 4. the desalination of dialysing obtains the solubility target protein.

4. method as claimed in claim 3 is characterized in that, described step is the host bacterium with intestinal bacteria (Escherichia coli) Rosetta-gami in 1., is carrier with the plasmid vector pET28a (+) that contains people SAA (hSAA1) gene order.

5. method as claimed in claim 3 is characterized in that, described step is removed 20 amino acid of the N end of SAA precursor protein in 1., and adds 6 affine polypeptide markers of poly Histidine at its N end.

6. method as claimed in claim 3 is characterized in that, 3. described step is by nickel affinity column single step purification.

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