CN102676533A - Recombinant human cystatin C coding gene and expression method - Google Patents
Recombinant human cystatin C coding gene and expression method Download PDFInfo
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- CN102676533A CN102676533A CN2012101516066A CN201210151606A CN102676533A CN 102676533 A CN102676533 A CN 102676533A CN 2012101516066 A CN2012101516066 A CN 2012101516066A CN 201210151606 A CN201210151606 A CN 201210151606A CN 102676533 A CN102676533 A CN 102676533A
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Abstract
The invention discloses a recombinant human cystatin C coding gene and an expression method. The nucleotide sequence of the coding gene is shown in SEQ ID NO:1, and the expression method of recombinant human cystatin C protein comprises the following steps that the recombinant human cystatin C coding gene is inserted into a polyclone locus of a prokaryotic expression carrier, a recombinant expression plasmid is constructed, the recombinant expression plasmid is converted to an expression host bacterium escherichia coli, and positive clone bacteria are screened out to obtain engineering bacteria; and the engineering bacteria are fermented, induced, separated and purified to obtain the recombinant human cystatin C protein. The recombinant human cystatin C coding gene provided by the invention can express the human cystatin C protein, the operation is convenient, the purity of the human cystatin C protein expressed by the coding gene reaches more than 90%, the expression efficiency is high, and no higher expression efficiency exists currently.
Description
Technical field
The invention belongs to biomedical engineering field, particularly a kind of recombinant human cystatin C encoding sox and expression method.
Background technology
Bladder chalone C (cystatin C, Cys C) is the L-Cysteine HCL Anhydrous arrestin, nineteen eighty-three Anastasi etc. first in Ovum Gallus domesticus album separation and purification obtain highly purified cystatin (cysteine proteinase inhibitor; CPI) be named as bladder chalone C after; Also being called as γ-trace of albumin and γ-postglobulin, extensively being present in the nucleated cell and body fluid of various tissues, is a kind of lower molecular weight, alkaline non-glycated proteins; Molecular weight is 13.3KD; Be made up of 122 amino-acid residues, can be produced by all nucleated cells of body, production rate is constant.Bladder chalone C in the circulation only is eliminated through glomerular filtration, is a kind of endogenous mark that reflects that glomerular filtration rate(GFR changes, and heavily absorbs at proximal convoluted tubule; But heavily absorb the back by metabolism decomposition fully, do not return blood, therefore; Its blood level is determined by glomerular filtration; And do not rely on any foeign element, and be a kind of desirable homology mark that reflects that glomerular filtration rate(GFR changes, can be used as the glomerular filtration functional parameter; Renal function test item commonly used at present has higher sensitivity and specificity, and it is to substitute the first-selected renal function evaluation index that traditional blood urine element, creatinine (Cr) and endogenous creatinine clearance rate (Ccr) are measured that its serum-concentration is measured.
Because the source of Cys C and antibody thereof is limited, the existing test kit that detects Cys C costs an arm and a leg, and has hindered it to apply at home.At present, adopt round pcr to transfer Cys C encoding sox usually, carry out recombinant expressedly behind the clone, but expression efficiency is extremely low, is difficult to satisfy market demand.We have designed the encoding sox of Cys C again for this reason, have set up the proteic preparation system of recombinant C ys C, in the hope of solving the source problem that comes of Cys C, for the research and development of CysC detection kit lay the foundation.
Summary of the invention
The object of the present invention is to provide a kind of recombinant human cystatin C encoding sox and expression method.
The technical scheme that the present invention taked is:
A kind of recombinant human cystatin C encoding sox, its nucleotide sequence is following:
5’-atgcgtggttctcatcatcaccatcaccacgctggtatcgaaggtcgttcctctccaggtaaaccgccacgtctggttggcggtccaatgg
atgcatccgtggaagaggaaggcgttcgtcgtgcactggactttgctgttggcgagtacaacaaagcgtccaatgacatgtatcactctcgtgctctgcaagtggttcgcgcacgcaagcagatcgtagcaggtgtgaactactttctggacgtagagctgggtcgcaccacttgcaccaaaacccagccgaatctggacaactgtccgttccatgaccaaccgcacctgaaacgcaaggcgttctgctcctttcagatctatgctgtaccatggcaaggcaccatgactctgtccaagtctacctgtcaggatgcgtaa-3’?（SEQ?ID?NO：1）。
The proteic expression method of recombinant human cystatin C comprises the steps:
1) the described recombinant human cystatin C encoding sox of claim 1 (goal gene) is inserted into the MCS of prokaryotic expression carrier; Make up recombinant expression plasmid; Recombinant expression plasmid is transformed into expressive host bacterium intestinal bacteria, filters out the positive colony bacterium, get engineering bacteria;
2) engineering bacteria through ferment, induce, separation, purifying, recombinant human cystatin C albumen.
Preferably, step 2) engineering bacteria is through fermentation culture, IPTG abduction delivering fusion rotein, collection thalline, and broken thalline is collected supernatant, and supernatant is analysed column purification through nickel dam, gets recombinant human cystatin C albumen.
Preferably, the method for IPTG abduction delivering fusion rotein: the bacterium liquid OD that treats fermentation culture
600nm0.6～1.0, add the IPTG of final concentration 0.1～10 mmol/L, cultivate 2～10 h for 30～40 ℃.
Preferably, the proteic expression method of recombinant human cystatin C comprises the steps:
1) two ends with goal gene add restriction enzyme site, carry out complete sequence and synthesize, and get synthetic gene sequence;
2) synthetic gene sequence is cloned the carrier in T, order-checking confirms successfully to make up reorganization T carrier;
3) goal gene is downcut from reorganization T carrier, be inserted into the MCS of prokaryotic expression carrier, make up recombinant expression plasmid, recombinant expression plasmid is transformed into expressive host bacterium intestinal bacteria, filter out the positive colony bacterium, get engineering bacteria;
4) engineering bacteria through ferment, induce, separation, purifying, recombinant human cystatin C albumen.
Preferably, the restriction enzyme site of goal gene two ends adding is inequality.Adding that different restriction enzyme site requirements do not exist in goal gene, is in order just with carrier to link to each other simultaneously, and the carrier that enzyme is cut self can not take place connects.
Preferably, the restriction enzyme site that its two ends add is not contained in the sequence inside of goal gene.
Preferably, the restriction enzyme site that the goal gene two ends add has identical restriction enzyme site with the prokaryotic expression carrier MCS, and enzyme is cut sequence consensus.
Preferably, 5 ' end of goal gene adds Nde I restriction enzyme site, and 3 ' end adds EcoR I restriction enzyme site; The T carrier is pMD18-T, and prokaryotic expression carrier is pET-22b (+); The expressive host bacterium is E.coli BL21 (DE3).
The design of a kind of recombinant human cystatin C encoding sox of the present invention is the aminoacid sequence according to human cystatin C; Tabulate according to colibacillary preference codon; The gene order that tabulation is formed according to codon; Carry out attributive analysises such as restriction enzyme site, GC content,, selected the encoding sox of recombinant human cystatin C of the present invention at last according to the expression amount of recombinant human cystatin C.
The principle of design of a kind of recombinant human cystatin C encoding sox of the present invention is: one group of amino acid that codon is corresponding unique, and the same amino acid of part can be by different ciphers coding; Each species all has the codon of some preferences simultaneously, therefore can under the situation that does not change coded aminoacid sequence, improve the efficient of the sequential structure raising proteins encoded of encoding sox through the codon of adjustment species preference.
The present invention at first utilizes the codon of intestinal bacteria preference to adjust coding gene sequence to improve the expression efficiency of target protein in intestinal bacteria, and restriction enzyme site, each nucleotide content etc. has been carried out adjustment to good grounds then gene order so that the genetically engineered operation.The adjustment of coding gene sequence not only can improve the expression efficiency of target protein recombinant human Cys C, also can make things convenient for the genetically engineered operation in the building process, has dual function.
The invention has the beneficial effects as follows:
Recombinant human cystatin C encoding sox provided by the invention, can expressing human bladder chalone C albumen, easy to operate, and the human cystatin C albumen of being expressed by encoding sox, purity reaches more than 90%, expression efficiency is high, does not see that at present expression efficiency better reports.Purification process of the present invention, the restriction that preparation condition and kind of carrier are selected is few, and the building process of expression vector is easy.
Description of drawings
Fig. 1 cuts evaluation figure for the enzyme of recombinant plasmid pET-CysC;
Fig. 2 is the Protein Detection figure of thalline behind the IPTG abduction delivering;
Fig. 3 is that the Western blotting of thalline behind the IPTG abduction delivering detects figure;
Fig. 4 is the Protein Detection figure of recombinant human cystatin C albumen affinity purification.
Embodiment
Embodiment below in conjunction with concrete is further described the present invention, but does not limit to so.
The proteic expression method of recombinant human cystatin C comprises the steps:
1) 5 ' of goal gene (nucleotides sequence is classified SEQ ID NO:1 as) is held adding Nde I restriction enzyme site (catatg), 3 ' end adds EcoR I restriction enzyme site (gaattc), carries out complete sequence and synthesizes, and gets synthetic gene sequence;
2) synthetic gene sequence is cloned the carrier in pMD18-T, order-checking confirms successfully to make up reorganization T carrier;
3) the correct goal gene that will check order downcuts from reorganization T carrier with Nde I and EcoR I restriction endonuclease; Purifying obtains goal gene, and prokaryotic expression carrier pET-22b (+) cuts processing through identical enzyme, the big segmental gene of purifying; And with double digestion after the goal gene that obtains be connected; Change competent cell DH5 α over to, the extracting plasmid gets recombinant expression plasmid pET-Cys C;
4) recombinant expression plasmid pET-Cys C is changed among the competence expressive host bacterium E.coli BL21 (DE3), coat on the LB solid medium (containing 100 mg/L penbritins), cultivate 20 h for 37 ℃, filtering out the positive colony bacterium is engineering bacteria;
5) engineering bacteria is seeded in the LB nutrient solution (containing 100 mg/L penbritins), 37 ℃ of cultivations are treated bacterium liquid OD600nm 0.8, add the IPTG of final concentration 1 mmol/L, cultivate 4 h for 37 ℃, and 4 ℃ of spinnings get abduction delivering supernatant and thalline;
6) with thalline with Binding Buffer washing 1 time after, carrying out ultrasonic bacteria breaking is centrifugal on ice; Collect supernatant,, fully dissolve inclusion body the deposition of the carrying out ultrasonic bacteria breaking after washing ice bath 1 h; Centrifugal, collect supernatant, the supernatant of twice collection is joined the nickel chromatography column; Add Binding Buffer successively, Wash Buffer, last Elute Buffer carries out the target protein wash-out; Collect Elute Buffer eluted protein, get recombinant human cystatin C albumen, adopt the purity of 12% SDS-PAGE electrophoresis detection target protein.
The proteic expression method of recombinant human cystatin C comprises the steps:
1) the recombinant expression plasmid pET-Cys C that embodiment 1 step 3) is obtained changes among the competence expressive host bacterium E.coli BL21 (DE3); Coat on the LB solid medium (containing 100 mg/L penbritins); Cultivate 16 h for 37 ℃, filtering out the positive colony bacterium is engineering bacteria;
2) engineering bacteria is seeded in the LB nutrient solution (containing 100 mg/L penbritins), bacterium liquid OD is treated in 37 ℃ of cultivations
600nm0.6, add the IPTG of final concentration 0.1 mmol/L, cultivate 10 h for 37 ℃, 4 ℃ of spinnings get thalline;
3) with embodiment 1 step 6).
The proteic expression method of recombinant human cystatin C comprises the steps:
1) the recombinant expression plasmid pET-Cys C that embodiment 1 step 3) is obtained changes among the competence expressive host bacterium E.coli BL21 (DE3); Coat on the LB solid medium (containing 100 mg/L penbritins); Cultivate 24 h for 37 ℃, filtering out the positive colony bacterium is engineering bacteria;
2) engineering bacteria is seeded in the LB nutrient solution (containing 100 mg/L penbritins), bacterium liquid OD is treated in 37 ℃ of cultivations
600nm1.0, add the IPTG of final concentration 10 mmol/L, cultivate 2 h for 37 ℃, 4 ℃ of spinnings get thalline;
3) with embodiment 1 step 6).
Comparative Examples 1
E.coli BL21 (DE3) is seeded in the LB nutrient solution, and 37 ℃ of cultivations are treated bacterium liquid OD600nm 0.8, add the IPTG of final concentration 1 mmol/L, cultivate 4 h for 37 ℃, and 4 ℃ of spinnings get thalline, and this thalline is the E.coli BL21 (DE3) after inducing.
Comparative Examples 2
PET-22b (+) plasmid is changed among the competence expressive host bacterium E.coli BL21 (DE3), coat on the LB solid medium (containing 100 mg/L penbritins), cultivate 20 h for 37 ℃, filtering out the positive colony bacterium is engineering bacteria; Engineering bacteria is seeded in the LB nutrient solution, and 37 ℃ of cultivations are treated bacterium liquid OD600nm 0.8, add the IPTG of final concentration 1 mmol/L, cultivate 4 h for 37 ℃, and 4 ℃ of spinnings get thalline, and this thalline is for transforming the E.coli BL21 (DE3) of pET-22b (+).
The recombinant expression pET-Cys C of embodiment 1 is adopted 1 respectively) Nde I single endonuclease digestion, 2) EcoR I single endonuclease digestion, 3) Nde I and EcoR I double digestion carry out enzyme and cut evaluation; Detect with 1% agarose gel electrophoresis, electrophorogram is seen Fig. 1, and mark 1 is DNA marker among Fig. 1; Mark 2 is that pET-Cys C is through Nde I single endonuclease digestion; Mark 3 be pET-Cys C through EcoR I single endonuclease digestion, mark 3 be pET-Cys C through Nde I and EcoR I double digestion, it is correct to obtain the purpose stripe size; Proof Cys C gene correctly is cloned into pET-22b (+) carrier, and the recombinant plasmid structure is correct.Recombinant plasmid pET-Cys C is checked order, and sequencing result is through the blast sequence alignment, and coded proteic aminoacid sequence and people Cys C mature peptide are in full accord.
Get the thalline (the E.coli BL21 (DE3) after inducing) that embodiment 1 step 5) induces the thalline that obtains (transforming the E.coli BL21 (DE3) of pET-Cys C), Comparative Examples 1 to induce to obtain and induce the three kinds of thalline of thalline (transforming the E.coli BL21 (DE3) of pET-22b (+)) that obtain with Comparative Examples 2; Adopt the 12%SDS-PAGE electrophoresis to carry out Protein Detection respectively; Electrophoresis detection result sees Fig. 2, and among Fig. 2, mark 1 is Comparative Examples 1 thalline; 2 is Comparative Examples 2 thalline; 3 and 4 is embodiment 1 thalline, and 5 is Marker, can be known by figure; The thalline that after inducing, transforms pET-Cys C contrasts bacterium has one obviously to increase thick protein band; Its relative molecular mass is (Cys C mature peptide is about 12kD, adds signal peptide, cleavage site and label polypeptide about 4kD) about 16kD, and the albumen of visible the inventive method abduction delivering is Cys C albumen.
The foregoing description 1 step 5) induces the thalline, the Comparative Examples 1 that obtain to induce the thalline and the Comparative Examples 2 that obtain to induce the thalline that obtains; Behind SDS-PAGE, change 75 volts with the electrotransfer appearance is wet respectively respectively, transferase 12 h is transferred to and carries out the western detection on the pvdf membrane; Skim-milk sealing transfer film; One anti-is the anti-people Cys of rabbit C polyclonal antibody (1: 1 000), and two anti-ly are goat-anti rabbit HRP-IgG (1: 3 000), and DAB is the colour generation substrate.Detected result is seen Fig. 3, and among the figure, 1 is Marker; 2 is Comparative Examples 1 thalline; 3 is Comparative Examples 2 thalline, and 4 is embodiment 1 thalline, can be known by figure; Can form the differential protein band with corresponding Cys C antibody behind the recombinant plasmid abduction delivering target protein, the albumen that proves abduction delivering of the present invention is Cys C albumen.
The abduction delivering supernatant that embodiment 1 step 5) is obtained, the thalline (being inclusion body) that step 5) obtains and the eluted protein behind the step 6) purifying; Adopt the purity of 12% SDS-PAGE electrophoresis detection target protein respectively; SDS-PAGE electrophoresis detection result sees Fig. 4, and annotate: 1 is Marker, and 2 is the abduction delivering supernatant; 3 is inclusion body, and 4,5 and 6 is the eluted protein behind the purifying.Can know by figure; Step 5) is separated the abduction delivering supernatant that obtains and is not contained Cys C albumen; Step 5) is separated the thalline obtain and is then contained step 5) and separate and obtain, and visible target protein exists for the master with the inclusion body form, behind the nickel chromatography column affinitive layer purification of the present invention; Purified product is single albumen, and target protein reaches more than 90% through gel imaging appearance scanning demonstration purity (does not see better bibliographical information of expression efficiency and publication) at present.
< 110>Guangzhou General Hospital Guangzhou Military Command
< 120>a kind of recombinant human cystatin C encoding sox and expression method
<130>
<160> 1
<170> PatentIn?version?3.5
<210> 1
<211> 411
<212> DNA
< 213>synthetic
<400> 1
atgcgtggtt?ctcatcatca?ccatcaccac?gctggtatcg?aaggtcgttc?ctctccaggt 60
aaaccgccac?gtctggttgg?cggtccaatg?gatgcatccg?tggaagagga?aggcgttcgt 120
cgtgcactgg?actttgctgt?tggcgagtac?aacaaagcgt?ccaatgacat?gtatcactct 180
cgtgctctgc?aagtggttcg?cgcacgcaag?cagatcgtag?caggtgtgaa?ctactttctg 240
gacgtagagc?tgggtcgcac?cacttgcacc?aaaacccagc?cgaatctgga?caactgtccg 300
ttccatgacc?aaccgcacct?gaaacgcaag?gcgttctgct?cctttcagat?ctatgctgta 360
ccatggcaag?gcaccatgac?tctgtccaag?tctacctgtc?aggatgcgta?a 411
Claims (9)
1. recombinant human cystatin C encoding sox, its nucleotide sequence is seen SEQ ID NO:1.
2. the proteic expression method of recombinant human cystatin C comprises the steps:
1) the described recombinant human cystatin C encoding sox of claim 1 (goal gene) is inserted into the MCS of prokaryotic expression carrier; Make up recombinant expression plasmid; Recombinant expression plasmid is transformed into expressive host bacterium intestinal bacteria, filters out the positive colony bacterium, get engineering bacteria;
2) engineering bacteria through ferment, induce, separation, purifying, recombinant human cystatin C albumen.
3. the proteic expression method of recombinant human cystatin C according to claim 2; It is characterized in that: step 2) engineering bacteria is through fermentation culture, IPTG abduction delivering fusion rotein, collection thalline, and broken thalline is collected supernatant; Supernatant is analysed column purification through nickel dam, gets recombinant human cystatin C albumen.
4. the proteic expression method of recombinant human cystatin C according to claim 3 is characterized in that: the method for IPTG abduction delivering fusion rotein: the bacterium liquid OD that treats fermentation culture
600nm0.6～1.0, add the IPTG of final concentration 0.1～10 mmol/L, cultivate 2～10 h for 30～40 ℃.
5. the proteic expression method of recombinant human cystatin C according to claim 2, it is characterized in that: this method comprises the steps:
1) two ends with goal gene add restriction enzyme site, carry out complete sequence and synthesize, and get synthetic gene sequence;
2) synthetic gene sequence is cloned the carrier in T, order-checking confirms successfully to make up reorganization T carrier;
3) goal gene is downcut from reorganization T carrier, be inserted into the MCS of prokaryotic expression carrier, make up recombinant expression plasmid, recombinant expression plasmid is transformed into expressive host bacterium intestinal bacteria, filter out the positive colony bacterium, get engineering bacteria;
4) engineering bacteria through ferment, induce, separation, purifying, recombinant human cystatin C albumen.
6. the proteic expression method of recombinant human cystatin C according to claim 5 is characterized in that: the restriction enzyme site that the goal gene two ends add is inequality.
7. the proteic expression method of recombinant human cystatin C according to claim 5 is characterized in that: the restriction enzyme site that its two ends add is not contained in the sequence inside of goal gene.
8. the proteic expression method of recombinant human cystatin C according to claim 5 is characterized in that: the restriction enzyme site that the goal gene two ends add has identical restriction enzyme site with the prokaryotic expression carrier MCS, and enzyme is cut sequence consensus.
9. according to the proteic expression method of each described recombinant human cystatin C of claim 5～8, it is characterized in that: 5 ' end of goal gene adds Nde I restriction enzyme site, and 3 ' end adds EcoR I restriction enzyme site; Prokaryotic expression carrier is pET-22b (+); The expressive host bacterium is E.coli BL21 (DE3).
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103435701A (en) * | 2013-07-24 | 2013-12-11 | 山西农业大学 | Pig antibacterial peptide cystatin11 fusion protein, and encoding gene and application thereof |
| CN106554411A (en) * | 2015-09-29 | 2017-04-05 | 北京九强生物技术股份有限公司 | Can be used as bladder chalone C product, the Its Preparation Method And Use of standard substance |
| CN109975552A (en) * | 2017-12-28 | 2019-07-05 | 江苏众红生物工程创药研究院有限公司 | A kind of recombination cystatin C albumen and its application in detection kit |
| CN110172434A (en) * | 2019-05-23 | 2019-08-27 | 华东理工大学 | A kind of genetic engineering bacterium producing human cystatin C and method |
-
2012
- 2012-05-16 CN CN2012101516066A patent/CN102676533A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| NCBI: "cystatin C [Homo sapiens]", 《GENBANK》 * |
| NCBI: "cystatin-C precursor [Homo sapiens]", 《GENBANK》 * |
| NCBI: "ompA - cystatin C fusion preprotein (AA -21 to 120) [synthetic construct]", 《GENBANK》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103435701A (en) * | 2013-07-24 | 2013-12-11 | 山西农业大学 | Pig antibacterial peptide cystatin11 fusion protein, and encoding gene and application thereof |
| CN103435701B (en) * | 2013-07-24 | 2014-09-17 | 山西农业大学 | Pig antibacterial peptide cystatin11 fusion protein, and encoding gene and application thereof |
| CN106554411A (en) * | 2015-09-29 | 2017-04-05 | 北京九强生物技术股份有限公司 | Can be used as bladder chalone C product, the Its Preparation Method And Use of standard substance |
| CN106554411B (en) * | 2015-09-29 | 2020-05-08 | 北京九强生物技术股份有限公司 | Cystatin C product capable of being used as standard substance, preparation method and application thereof |
| CN109975552A (en) * | 2017-12-28 | 2019-07-05 | 江苏众红生物工程创药研究院有限公司 | A kind of recombination cystatin C albumen and its application in detection kit |
| CN110172434A (en) * | 2019-05-23 | 2019-08-27 | 华东理工大学 | A kind of genetic engineering bacterium producing human cystatin C and method |
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Application publication date: 20120919 |