CN101899457A - Recombinant carp GLS gene, protein and its preparation and detection method and application - Google Patents
Recombinant carp GLS gene, protein and its preparation and detection method and application Download PDFInfo
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Abstract
The invention discloses a recombinant carp GLS gene, a recombinant carp GLS protein, preparation methods, detection methods and application thereof. The recombinant carp GLS gene is shown by SEQ ID NO:1. The amino acid sequence expressed by the recombinant carp GLS gene is shown by SEQ ID NO:2. The research and the development on gene cloning associated with the carp GLS protein and recombinant expression thereof have extremely important effect, because clear research on the functions of the carp GLS gene and the carp GLS protein can be used for improving the survival rate of the cell culture, controlling metabolism utilization of GLn by a cell to regulate and control the cell growth and the like, and an antibody prepared from the recombinant GLS protein can detect the expression condition of the GLS gene in various cells, different cell cycles, and different developmental stages of individual cells.
Description
Technical field
The present invention relates to a kind of reorganization carp GLS gene, albumen and preparation and detection method and application.
Background technology
(Glutaminase is that (catalysis Gln hydrolysis generates L-glutamic acid and ammonia to glutamine for Glutamine, Gln) key enzyme and the rate-limiting enzyme of metabolism utilization GLS) to L-Glutamine deaminase.Mammiferous GLS is divided into kidney (K-GLS) and two kinds of hypotypes of liver (L-GLS), and only there is K-GLS in enteron aisle.5 ' of rat K-GLS gene cDNA partial sequence-hold non-translational region into 60bp, the 2022bp open reading frame, 3 '-hold non-translational region into 2445bp; 674 amino-acid residues of open reading frame coding, molecular weight is the precursor protein of 74kDa, comprises 16 amino-acid residues of N-end, forms the plastosome positioning sequence of typical amphiphilic α-Luo Xuanjiegou; Form 66kDa GLS subunit since 73～90 amino-acid residues; GLS mRNA 3 '-end non-translational region comprises two AAUAAA polyadenylation signals.
GLS is organism metabolism and the key enzyme that utilizes Gln, and its active height plays important regulation at growth and proliferation of cell in the differentiation and maturation process.The activity that suppresses GLS causes body's immunological function to descend, the intestinal mucosa dysfunction, and digestion and absorptive function are impaired.Body can reduce enteron aisle GLS activity under the disease situation, thereby reduces the metabolism utilization of enteron aisle to Gln, and it is normal to influence the enteron aisle structure.Under the Gln shortage situation, the GLS enzymic activity of enteron aisle reduces, but GLS mRNA abundance increases, and to strengthen under the nutrition rehabilitation normal circumstances, enteron aisle is to metabolism and the utilization of Gln.Daily ration adds Gln can improve the activity of enteron aisle GLS and the abundance of GLS mRNA.
So far, research about GLS performance regulating effect in mammalian cell growth and propagation is carried out in mammiferous cells such as people, rat, mouse, and many achievements have been obtained, but the relevant low research report that waits GLS in the vertebrates fish cell of Shang Weijian, do not see yet cDNA nucleotide sequence that carp GLS gene is arranged and with the report of its corresponding amino acid sequence.
Summary of the invention
Technical problem to be solved by this invention provides a kind of reorganization carp GLS gene, albumen and preparation and detection method and application.
The present invention addresses the above problem the technical scheme that is adopted: the reorganization carp GLS gene shown in a kind of SEQ of having ID NO:1.
A kind of have shown in the SEQ ID NO:2 by the carp GLS gene expressed aminoacid sequence of reorganization shown in the SEQ ID NO:1.
The preparation method of the reorganization carp GLS gene shown in a kind of SEQ ID NO:1 comprises the steps:
1) gathers total RNA of carp and be that template is carried out the reverse transcription operation, obtain cDNA first chain with the total RNA of its intestinal tissue;
2) be template with first chain of the cDNA in the step 1), utilize primer G1, G2 to carry out pcr amplification, obtain the sequence shown in the SEQ ID NO:1, the sequence of described G1, G2 is as follows respectively:
G1：5’-ATGTTACACTTTAGGCTTTCGAGG-3’，
G2：5’-CTACAGCATCCCGTCAAGGTTTTTC-3’。
Described step 2) is primer with G1, G2 in, utilizes archaeal dna polymerase to carry out in the 50 μ l pcr amplification systems of PCR reaction each component and ratio is: cDNA first chain, 2.5 μ l; The G1 of 50 μ M, 0.25 μ l; The G2 of 50 μ M, 0.25 μ l; 10 * LA PCR Buffer II (Mg
2+Plus), 5.0 μ l; The dNTP Mixture of each 2.5mM, 8.0 μ l; DdH
2O, 33.5 μ l; The archaeal dna polymerase of 5U/ μ l, 0.5 μ l; Reaction cycle condition: 94 ℃ of pre-sex change 1min; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ are extended 2min, totally 35 circulations; Last 72 ℃ are extended 8min, and reaction is finished.
A kind of detection method of reorganization carp GLS gene of method for preparing comprises the steps:
1) pcr amplification with G1 and G2PCR recovery product adopts 2 * Taq PCR MasterMix to carry out, and each component and ratio thereof are in the 50 μ l pcr amplification systems: the GLS of 50 times of dilutions reclaims product, 1.0 μ l; The G3 of 50 μ M, 0.25 μ l; The G4 of 50 μ M, 0.25 μ l; 2 * Taq PCR MasterMix, 25.0 μ l; DdH
2O, 23.5 μ l; Reaction cycle condition: 94 ℃ of pre-sex change 1min; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ are extended 1min 30sec, totally 32 circulations; Last 72 ℃ are extended 5min, and reaction is finished; The sequence of described G3, G4 is as follows respectively:
Upstream primer G3,5 '-GCCAGGTAGCAGATTACATTCC-3 ',
Downstream primer G4,5 '-GTGATTGGACAGAAGCCGCC-3 ';
2) get 5 μ l PCR products with 1.0% sepharose at 100V, carry out electrophoresis detection under the condition of 15min, gel imaging system observation analysis result.
A kind of detection method of reorganization carp GLS gene of method for preparing comprises the steps:
1) pcr amplification with G1 and G2PCR recovery product adopts 2 * Taq PCR Master Mix to carry out, and each component and ratio thereof are in the 50 μ l pcr amplification systems: the GLS of 50 times of dilutions reclaims product, 1.0 μ l; The G5 of 50 μ M, 0.25 μ l; The G6 of 50 μ M, 0.25 μ l; 2 * Taq PCR Master Mix, 25.0 μ l; DdH
2O, 23.5 μ l; Reaction cycle condition: 94 ℃ of pre-sex change 1min; 94 ℃ of sex change 30sec, 56.5 ℃ (G5G6) 30sec that anneals, 72 ℃ are extended 1min 30sec, totally 32 circulations; Last 72 ℃ are extended 5min, and reaction is finished; The sequence of described G5, G6 is as follows respectively:
Upstream primer G5,5 '-ACCTCTGGGCTGTTTCTCTC-3 ',
Downstream primer G6,5 '-AACTGGATTCCCCTCACGCT-3 ';
2) get 5 μ l PCR products with 1.0% sepharose at 100V, carry out electrophoresis detection under the condition of 15min, gel imaging system observation analysis result.
In the above-mentioned detection method, the gel of 50 times of dilutions reclaims the product prepared reorganization carp GLS gene cDNA sequence among the preparation method of carp GLS gene cDNA encoding region nucleotide sequence that refers to recombinate.
The application of above-mentioned reorganization carp GLS gene in detecting carp GLS expression of gene situation comprises that carp GLS gene is at various types of cells and tissue, body early embryo, individual expression under different states.
In detection, according to sequences Design following primer shown in the SEQ ID NO:1:
Upstream primer GD1,5 '-GCTGTGTTCCATTGAGGTGAC-3 ';
Downstream primer GD2,5 '-ACTCGCTCGCCTGTGATTG-3 ';
Adopt fluorescent quantitative PCR, each component and ratio thereof are in the 20 μ l pcr amplification systems: cDNA, 2.0 μ l; The GD1 of 10 μ M, 1.0 μ l; The GD2 of 10 μ M, 1.0 μ l; SYBR
Premix Ex Taq
TMII (2 *), 10.0 μ l; DH
2O, 6.0 μ l; Reaction cycle condition: 95 ℃ of pre-sex change 30sec; 95 ℃ of sex change 5sec, 55 ℃ of annealing 15sec, 72 ℃ are extended 15sec, totally 44 circulations; Last 72 ℃ are extended 2min, and reaction is finished.
The gene clone that the present invention research and development is relevant with carp GLS albumen and recombinant expressedly have an important role, because GLS gene and the proteic function of carp are known in research, can be used in raising cell cultures survival rate, control the metabolism utilization of cell Gln, with aspects such as regulating cell growths, can detect the expression of GLS gene in different types of cell, different cell cycle and individual different developmental phases by the antibody of reorganization GLS protein Preparation.
The present invention separates the GLS gene from the different tissues cell of carp.As a result of, the inventor has separated the cDNA coding region 1788bp fragment of GLS gene from carp enteron aisle histocyte, and has determined its nucleotide sequence and the aminoacid sequence of inferring thus.The inventor is by the known zebra fish of comparison, the people, rat, the nucleotide sequence of the GLS gene of mouse, find conserved regions, then according to the sequence of GLS gene (NM_001045044) the cDNA coding region of zebrafish embryo, design upstream primer (5 '-from initiator codon ATG's) under the prerequisite of amino acid coding not breaking, downstream primer 5 ' end originates in the natural terminator codon TGA of zebra fish GLS gene, designed and a pair ofly be used for that (upstream primer is called G1 by the segmental primer in RT-PCR method amplification carp GLS gene cDNA encoding district, downstream primer is called G2), and use this primer amplification has been gone out specific fragment, obtained the cDNA coding region 1788bp fragment of carp GLS gene after the order-checking, sequential analysis shows that sequence (NM_001045044) homology with zebra fish is 89.9%, and kept correct coding, the aminoacid sequence of the sequence encoding of deriving is thus compared with the total number of atnino acid of zebra fish sequence and is only differed 4 amino-acid residues.This cDNA fragment is called " cGLS ".
So, the objective of the invention is nucleotide sequence design primer by the GLS of known different plant species, clone the cDNA coding region fragment of carp GLS gene then by the RT-PCR method.To the nucleotide sequence of the coding region 1788bp of cDNA that coding carp GLS proteic gene is provided behind this sequencing fragment and the aminoacid sequence (sequence sees sequence table for details) of deduction thus.
In sum, the invention has the beneficial effects as follows: the gene clone that the present invention research and development is relevant with carp GLS albumen and recombinant expressedly have an important role, because GLS gene and the proteic function of carp are known in research, can be used in raising cell cultures survival rate, control the metabolism utilization of cell Gln, with aspects such as regulating cell growths, can detect the expression of GLS gene in different types of cell, different cell cycle and individual different developmental phases by the antibody of reorganization GLS protein Preparation.
Description of drawings
Fig. 1 is the electrophorogram from the result of the RT-PCR of total RNA of Intestinum Cyprinus carpio cellular segregation (the cDNA cGLS of 1788bp);
Fig. 2 is template with G1 and G2 primer to amplified production, is primer or is that primer carries out the electrophorogram that PCR identifies with G5, G6 with G3, G4.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
The aminoacid sequence of reorganization of the present invention carp GLS gene cDNA encoding region nucleotide sequence and expressing protein thereof respectively as: shown in SEQ ID NO:1, the SEQ ID NO:2.
In order to separate the cGLS gene from the carp histocyte, the inventor at first gathers all kinds of tissue samples of carp (Common carp, Cyprinus carpio) according to the standard-required that extracts total RNA.Tissue block such as kidney, enteron aisle, muscle, spleen and liver pancreas are got after slaughtering carp immediately in the scene, put into to go into liquid nitrogen behind the freeze pipe immediately and preserve, and take back the laboratory and be placed on-80 ℃ of refrigerators to preserve standby.Utilize the total RNA extraction reagent box (TaKaRa RNAiso Reagent) of TaKaRa then and extract total RNA that carp enteron aisle, muscle, kidney, liver pancreas and spleen etc. are organized according to the schedule of operation of working instructions, the expression of Mammals GLS gene in all kinds of tissues and total RNA according to bibliographical information extracts the result at last, selected is that template is carried out the reverse transcription operation with the total RNA of intestinal tissue, obtains cDNA first chain.Be template with this cDNA first chain again, utilize Auele Specific Primer G1, G2 to carry out pcr amplification, obtain the purpose fragment.Afterwards isolating purpose fragment behind the electrophoresis is cut glue and reclaim, measure the concentration of double-stranded cDNA, amplified production is identified with nested PCR method.The right amplified production of G1 and G2 primer is by the pcr amplification of G3 and G4, G5 and G6, and electrophoresis is identified obtains expecting the dna fragmentation of size, preliminary identification the exactness of G1 and G2 amplified production.The sample of the amplified production that G1 and G2 primer is right is served the sea and is given birth to the order-checking of worker's biotechnology company limited.As a result of, obtained the nucleotide sequence in the carp cGLS gene cDNA encoding district of 1788bp.
The specific PCR primer G1 of the present invention and the G2 that show feature above-mentioned can utilize the RT-PCR method to amplify the cDNA coding region fragment of the cGLS gene of carp.The nucleotide sequence of the carp GLS gene cDNA that obtains according to the present invention can further detect the tissue expression specificity of GLS gene, the clone that also can further realize carp GLS full length gene cDNA by the method for RT-PCR or hybridization.CDNA of the present invention recombinated may give expression to complete GLS albumen on the expression vector, and then can be used for antibody producing, detects carp various types of cells and tissue, body early embryo, individual GLS expression of gene situation under different states or the like.
The segmental clone of cDNA coding region 1788bp of carp GLS gene
In order to clone the cDNA that comprises the 1788bp coding region from the histiocytic GLS gene of carp enteron aisle, according to disclosed nucleotide sequence (NM_001045044, NM_001031248, NM_001081081, NM_014905), at first the PCR primer of the cDNA of the synthetic permission amplification of design 1788bp is right, that is:
Upstream primer 5 '-ATGTTACACTTTAGGCTTTCGAGG-3 ' (G1)
Downstream primer 5 '-CTACAGCATCCCGTCAAGGTTTTTC-3 ' (G2).
Other two pairs of primers that are used to differentiate G1 and G2 amplified production have been synthesized in design then, that is:
Upstream primer G3,5 '-GCCAGGTAGCAGATTACATTCC-3 ',
Downstream primer G4,5 '-GTGATTGGACAGAAGCCGCC-3 ';
Upstream primer G5,5 '-ACCTCTGGGCTGTTTCTCTC-3 ',
Downstream primer G6,5 '-AACTGGATTCCCCTCACGCT-3 '.
In order to utilize the cDNA of RT-PCR method clone GLS gene, from the total RNA of carp enteron aisle tissue extraction, utilize TaKaRa total RNA extraction reagent box (TaKaRa RNAiso Reagent) and extract carp enteron aisle total tissue RNA, carry out electrophoresis detection and ultraviolet determination RNA concentration and be placed on-80 ℃ of refrigerators and preserve standby according to the schedule of operation of working instructions.Then, utilize the M-MLV ThermoScript II of TaKaRa and carry out reverse transcription reaction, obtain cDNA first chain according to the schedule of operation of working instructions.
With cDNA first chain that obtains above is template, and G1, G2 are primer, utilizes TaKaRa LA Taq archaeal dna polymerase to carry out the PCR reaction.50 μ l pcr amplification system: cDNA, 2.5 μ l; G1 (50 μ M), 0.25 μ l; G2 (50 μ M), 0.25 μ l; 10 * LA PCR Buffer II (Mg
2+Plus), 5.0 μ l; DNTP Mixture (each 2.5mM), 8.0 μ l; DdH
2O, 33.5 μ l; TaKaRa LA Taq (5U/ μ l), 0.5 μ l.Reaction cycle condition: 94 ℃ of pre-sex change 1min; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ are extended 2min, totally 35 circulations; Last 72 ℃ are extended 8min, and reaction is finished.Get 5 μ l PCR products with 1.0% sepharose carry out electrophoresis detection (100V, 15min), gel imaging system observation analysis result.As a result of, obtained the specificity purpose fragment (sequence shown in the SEQ ID NO:1) of 1788bp.
The pcr amplification that G1 and G2PCR reclaim product adopts 2 * Taq PCR Master Mix (KT201) to carry out, and 50 μ l pcr amplification system: GLS reclaim product (50 * dilution), 1.0 μ l; G3 (50 μ M), 0.25 μ l and G4 (50 μ M) or G5 (50 μ M), 0.25 μ l and G6 (50 μ M), 0.25 μ l; 2 * TaqPCR Master Mix, 25.0 μ l; DdH
2O, 23.5 μ l.Reaction cycle condition: 94 ℃ of pre-sex change 1min; 94 ℃ of sex change 30sec, 55 ℃ (G3G4) or 56.5 ℃ (G5G6) 30sec that anneals, 72 ℃ are extended 1min 30sec, totally 32 circulations; Last 72 ℃ are extended 5min, and reaction is finished.Get 5 μ l PCR products with 1.0% sepharose carry out electrophoresis detection (100V, 15min), gel imaging system observation analysis result.As a result of, obtained the amplified production of about 600 (Fig. 2) and about 800bp (Fig. 2) respectively.Its size conforms to the amplified production 820bp of G5G6 primer to design with the amplified production 598bp of G3G4 primer to design, shows the specificity of amplified production.
G1G2 amplified production through twice evaluation is sent to Shanghai living worker's biotechnology company limited with checking order.As a result of, obtained the nucleotide sequence (Fig. 2) of cDNA coding region of the carp GLS gene of 1788bp.
The nucleotide sequence of the cDNA of carp GLS gene
SEQ ID NO:1 has shown the cDNA that this embodiment obtained clone's nucleotide sequence, and SEQ IDNO:2 is the aminoacid sequence of inferring thus.The nucleotide sequence of GLS gene, it has comprised the nucleotide sequence of the 1788bp of cDNA coding region, 595 amino-acid residues of the mature protein of the 65.96KDa that coding formation molecular weight is inferred.
The comparison of the nucleotide sequence of the nucleotide sequence of the reorganization carp GLS gene cDNA that the present invention obtained and zebra fish (NM_001045044), jungle fowl (NM_001031248), mouse (NM_001081081) and people's (NM_014905) GLS gene cDNA.The result shows: 1) nucleotide sequence of the nucleotide sequence of carp GLS gene cDNA and zebra fish, jungle fowl, mouse and people's GLS gene cDNA has 89.9,69.4,70.2 and 69.8% similarity respectively.Aminoacid sequence of being derived by this section nucleotide sequence of carp and zebra fish, jungle fowl, mouse and people's similarity are respectively: 94.2,74.5,78.4 and 78.5%.
As clearly demonstrate and as above explanation, the invention provides the cDNA and the aminoacid sequence of deduction thus of the proteinic 1788bp of GLS of the primer that utilizes RT-PCR method clone carp GLS gene cDNA encoding district and coding carp.This cDNA has comprised the nucleotide sequence of 1788bp, and its coding contains the protein of 595 amino-acid residues, through further transform the back it can subclone to as expressing on protokaryon such as pET or pcDNA3.1 or the carrier for expression of eukaryon.The cDNA fragment of reorganization may give expression to complete GLS protein polypeptide section, and then can be used for antibody producing, detects carp various types of cells and tissue, body early embryo, individual GLS expression of gene situation under different states or the like.
The different intestinal segment GLS of carp genetic expression
This example is with the expression of fluorescent quantitative PCR technique research GLS gene at each intestinal segment of carp.Choose the healthy carp sampling of 6 approaching tails of body weight.Test fish enteron aisle is divided into 7 almost isometric intestinal segments according to the method for (1997) such as Villanueva, measures each intestinal segment GLS mRNA abundance respectively.
The sample liquid nitrogen grinding is extracted total RNA.Fluorescent quantitation adopts PrimeScript with carp enteron aisle cDNA first chain
TMRT reagent Kit (precious biotechnology (Dalian) company limited, DRR037AS) synthetic, the method operation of pressing the test kit specification sheets.
The amplification of GLS cDNA fluorescent quantitation: the cDNA coding region sequence of the reorganization carp GLS gene that the clone obtains according to the present invention is designed to a pair of GLS specificity quantitative amplification primer:
Upstream primer GD1,5 '-GCTGTGTTCCATTGAGGTGAC-3 ';
Downstream primer GD2,5 '-ACTCGCTCGCCTGTGATTG-3 '.
Fluorescent quantitative PCR adopts SYBR
Prime Script
TM(precious biotechnology (Dalian) company limited DRR083A) carries out RT-PCR Kit II (Perfect RealTime), 20 μ l pcr amplification system: cDNA, 2.0 μ l; GD1 (10 μ M), 1.0 μ l; GD2 (10 μ M), 1.0 μ l; SYBR
Premix ExTaq
TMII (2 *), 10.0 μ l; DH
2O, 6.0 μ l.Reaction cycle condition: 95 ℃ of pre-sex change 30sec; 95 ℃ of sex change 5sec, 55 ℃ of annealing 15sec, 72 ℃ are extended 15sec, totally 44 circulations; Last 72 ℃ are extended 2min, and reaction is finished.
The result shows: there is significant difference in the different intestinal segment GLS of carp mRNA relative expression quantity.Wherein: 3～4 intestinal segment mRNA relative expression quantities are significantly higher than 7 intestinal segments (P＜0.05); All the other each intestinal segment differences are remarkable (P＞0.05) not.
The different intestinal segment GLS of table 1 carp mRNA relative expression quantity (%, n=6)
Broken-hearted mRNA
1 1.04±0.24
b
2 0.96±0.21a
b
3 1.17±0.29
b
4 1.22±0.25
b
5 0.97±0.23
ab
6 1.00±0.23
ab
7 0.71±0.17
a
The identical expression statistical discrepancy of same column letter is not remarkable, and is alphabetical identical as institute's mark, is a or is b or is ab, represent relatively there was no significant difference of these two groups of data, such as mark letter inequality, as being labeled as a, ab respectively, represent that these two groups of data have significant difference (P＜0.05).
By detecting carp GLS gene at various types of cells and tissue, body early embryo, individual expression under different states, can the intermediate data result who detect further be analyzed and study, shape and correlation circumstance to carp GLS expression of gene situation, its expressing protein, do further research, and then can be used for antibody producing.
As mentioned above, just can realize the present invention preferably.
Sequence table
Claims (8)
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| 《中国优秀硕士学位论文全文数据库》 20091115 付保忠 鲤鱼蛋白酶体激活因子PA28-beta亚单位基因克隆、鉴定及时空转录分析 全文 1-8 , * |
| 《中国优秀硕士学位论文全文数据库》 20091115 付保忠 鲤鱼蛋白酶体激活因子PA28-beta亚单位基因克隆、鉴定及时空转录分析 全文 1-8 , 2 * |
| 付保忠: "鲤鱼蛋白酶体激活因子PA28-β亚单位基因克隆、鉴定及时空转录分析", 《中国优秀硕士学位论文全文数据库》 * |
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| CN105175524A (en) * | 2015-10-14 | 2015-12-23 | 四川农业大学 | Carp recombined antimicrobial protein and preparing method thereof |
| CN105175524B (en) * | 2015-10-14 | 2019-02-26 | 四川农业大学 | A kind of carp recombinant bacteriostatic protein and preparation method thereof |
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