CN101544692B - Rousettus leschenaulti leptin and its cDNA sequence - Google Patents
Rousettus leschenaulti leptin and its cDNA sequence Download PDFInfo
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- CN101544692B CN101544692B CN2009100394407A CN200910039440A CN101544692B CN 101544692 B CN101544692 B CN 101544692B CN 2009100394407 A CN2009100394407 A CN 2009100394407A CN 200910039440 A CN200910039440 A CN 200910039440A CN 101544692 B CN101544692 B CN 101544692B
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- leptin
- rousettus
- leu
- leschenaulti
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Abstract
The present invention discloses rousettus leschenaulti leptin and its cDNA sequence; the leptin is a sort of protein with an amino acid sequence shown in SEQ ID NO: 3, or protein with an amino acid sequence formed by deleting, displacing, or adding one or more pieces of amino acid in amino acid sequence (i), with the same function as the amino acid sequence (i). The present invention employs GST recombined expression technique and thrombosin excision technique to remove GST marker, to extract leptin from rousettus leschenaulti. The result of cell activity analysis indicates the rousettus leschenaulti leptin has obvious fat degradation activity, and can be used to prepare weight-reducing medicines, diabetes mellitus treatment medicines, hypertension treatment medicines, and fatty liver treatment medicines, etc.
Description
Technical field
The present invention relates to leptin protein, be specifically related to a kind of leptin protein and cDNA sequence thereof that derives from brown flying fox (Rousettusleschenaultii).
Background technology
Leptin protein (leptin) is a small protein of being made up of 167 amino acid of finding in 1994, by fatty tissue excretory protein hormone, at present verified other of result of study organized also and can be produced, and comprises stomach, the people's of skeletal muscle, the rat of placenta, rat the adenohypophysis etc. of mammary epithelial cell, people and mouse.
Leptin protein is fat relevant signal, and the feedback signal of fatty tissue affacts the brain center, and keep on a diet behavior and activity (metabolism and motion) are the products of ob (obese) gene.The leptin protein that fatty tissue produces is transported to brain through blood, and takes in by keeping on a diet behind the hypothalamic receptor acting and energy expenditure is controlled mammiferous body weight, is considered to one of slimming medicine of potentialization.
Leptin protein is except reducing appetite, management of body weight, reduce outside the body fat content, also can transmit the inside and outside energy storage signal of animal body to big mesencephalic centre, the endocrine system that affects the nerves promotes the animal reproductive system to grow and obtains Fertility, the reproduction activities such as startup, sexual maturity and gestation of regulation and control animal puberty, regulate fetal placenta, animal is oestrused in advance, shorten the oestrus cycle, regulate glucose metabolism, lipid metabolism, blood pressure regulation, brain and skeleton development, wound healing, cytodifferentiation propagation, participate in hemoposieis, functions such as immunity of organism adjusting.The leptin function diversity is the keying action aspect capacity control especially, also gives the value of the bigger product development of leptin.
The development and utilization of leptin protein has been deep into many fields and has comprised the exploitation of slimming medicine and the deep processing of diet products, the exploitation of treatment diabetes medicament, the exploitation of treatment hypertension drug, treatment fatty liver drug development or the like, along with to the deepening continuously of its function understanding, will be more wide about the leptin industrialization prospect.
Summary of the invention
The object of the present invention is to provide a kind of leptin protein that derives from brown flying fox (Rousettus leschenaultii), establish basic substance for developing the leptin gene engineering product from now on.
Another object of the present invention is to provide the cDNA that contains above-mentioned leptin protein complete coding region sequence.
Above-mentioned purpose of the present invention is achieved by following scheme:
The invention provides a kind of leptin protein, this leptin protein is following (i) or (ii) described each protein:
(i) a kind of protein with aminoacid sequence shown in the SEQ ID NO:3;
(ii) in aminoacid sequence (i), formed, and had the protein of identical function with the protein of aminoacid sequence (i) by the aminoacid sequence of disappearance, replacement or 1 of addition or several amino acid.
Leptin protein of the present invention preferably has the aminoacid sequence shown in the SEQ ID NO:3.
The present invention also provides the cDNA sequence of the above-mentioned leptin protein of coding, and this cDNA sequence is coding following (i) or (ii) described each proteinic cDNA sequence:
(i) a kind of protein with the aminoacid sequence shown in the SEQ ID NO:3;
(ii) in aminoacid sequence (i), formed, and had the protein of identical function with the protein of aminoacid sequence (i) by the aminoacid sequence of disappearance, replacement or 1 of addition or several amino acid.
Its nucleotide sequence of cDNA sequence preference of above-mentioned coding leptin protein is shown in SEQ ID NO:1.
Leptin protein of the present invention comes from brown flying fox (Rousettus leschenaultii), extract after the total RNA reverse transcription of brown flying fox fatty tissue as template, carry out the degenerate pcr amplification according to known Mammals leptin protein sequence conserved regions design degenerated primer, obtain the full length cDNA sequence of leptin protein of the present invention after the order-checking, and this full length cDNA sequence carried out amino acid analysis, obtaining Rousettus leschenaulti leptin of the present invention is the aminoacid sequence shown in the SEQ ID NO:3,165 amino acid altogether, wherein preceding 21 amino acid are signal peptide sequence.
Compared with prior art, the present invention has following advantage:
The present invention has obtained a kind of full-length cDNA of leptin protein from the fatty tissue of brown flying fox, and carried out protein expression, remove the GST marker by utilizing the excision of GST recombination and expression techniques and zymoplasm enzyme, successfully obtained Rousettus leschenaulti leptin, in addition this albumen is carried out the cytoactive analysis, the result shows that Rousettus leschenaulti leptin of the present invention has tangible fat acid decomposition activity, can be used for preparing slimming medicine, treatment diabetes medicament, treatment hypertension drug and treatment fatty liver medicine etc.
Description of drawings
Fig. 1 is the pcr amplification electrophorogram of Rousettus leschenaulti leptin full length cDNA sequence;
Wherein, 1 is molecular weight marker, and 2 is pcr amplification product;
Fig. 2 is that the SDS-PAGE of expression of recombinant plasmid product among the embodiment 2 detects electrophorogram;
Wherein, 1 is molecular weight marker, and 2 do not induce control sample for recombinant expressed bacterium, and 3 for recombinant expressed bacterium IPTG induces sample behind the 3h, and arrow indication place is new band of expression;
Fig. 3 is that the SDS-PAGE of protein expression among the embodiment 3 detects electrophorogram;
Wherein, 1 is molecular weight marker, and 2 were column purification but cut control sample without zymoplasm, and 3 was column purification and the sample cut through zymoplasm, and 4 is GST, and 5 is leptin protein;
Fig. 4 is the active detected result histogram of Rousettus leschenaulti leptin MTT;
Wherein, 1 is the GST control group, and 2 is the Rousettus leschenaulti leptin group, and 3 is the blank group,
*: P＜0.01;
Fig. 5 is the active detected result histogram of Rousettus leschenaulti leptin LDH;
Wherein, 1 is the GST control group, and 2 is the Rousettus leschenaulti leptin group, and 3 is the blank group,
*: P＜0.01.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but specific embodiment is not done any qualification to the present invention.
The acquisition of embodiment 1 Rousettus leschenaulti leptin full length cDNA sequence
1, template preparation
Extracting the total RNA of brown flying fox (Rousettusleschenaultii) fatty tissue according to RNAiso test kit (TakaRa company) specification sheets, and adopt the reverse transcription test kit, is template with this total RNA, carries out the reverse transcription test and obtains following pcr amplification template.
2, design of primers
According to known Mammals leptin protein sequence conserved regions design degenerated primer, as follows:
3, pcr amplification
Pcr amplification reaction system (50 μ l): pcr amplification template 2 μ l, each 1 μ l of primer Primer 1/Primer 2 (10pM), 10 * Taq Polymerase mix (TaKaRa company), 5 μ l, ddH
2O 41 μ l;
The pcr amplification reaction condition: 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 1min, 54 ℃ of annealing 90s, 72 ℃ are extended 2min, 30 circulations, last 72 ℃ are extended 5min.
4, the connection of PCR product, conversion and order-checking
Above-mentioned PCR reaction product is gone into pMD-19T carrier (commercially available) through routine operation rear clones such as glue recovery, connection, conversions, and positive colony send order-checking company to check order after blue hickie screening and PCR evaluation, simultaneously sequencing result is carried out amino acid sequence analysis.
Sequencing result and amino acid sequence analysis result show, the full-length cDNA of this enforcement amplification gained Rousettus leschenaulti leptin is 498bp, its nucleotide sequence is shown in SEQ ID NO:1,165 amino acid codings of this cDNA sequence encoding, its aminoacid sequence is shown in SEQ ID NO:3, and wherein preceding 21 amino acid are signal peptide sequence.
The structure of embodiment 2 recombinant expression vectors
According to embodiment 1 gained Rousettus leschenaulti leptin gene order design primer (P1 and P2), amplification does not contain whole coding regions of signal peptide sequence, and introduces the structure that BamH I/Sal I restriction site is used for recombinant expression vector.
P1, its nucleotide sequence is shown in SEQ ID NO:6;
P2, its nucleotide sequence is shown in SEQ ID NO:7.
Above-mentioned pcr amplification reaction system can be with reference to PCR reaction kit specification sheets, and its concrete reaction conditions is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 1min, 55 ℃ of annealing 90s, 72 ℃ are extended 2min, 30 circulations, last 72 ℃ are extended 5min.Amplified production can be seen purpose fragment amplification band clearly through 2% agarose gel electrophoresis, and as shown in Figure 1, the about 435bp of the fragment length that amplifies conforms to the expanding fragment length of design, and non-specific amplification do not occur.
With above-mentioned pcr amplification product after glue purification reclaims, spend the night in 37 ℃ of digestion with BamH I and Sal I, be connected with PGEX-4T-2 carrier (Invitrogen) subsequently and make up recombinant expression plasmid, selecting positive colony checks order, sequencing result is identical with former sequence, show that the gene behind the amplification clone is not undergone mutation, and open reading frame (ORF) is consistent with carrier ORF, about 373 amino acid of fusion rotein (containing GST), theoretical molecular are 41.8kDa.
Change in the escherichia coli expression bacterium BL21 (commercially available) through the correct positive colony of sequence verification sequence reading frame above-mentioned, (IPTG) induces 3h through the 1mmol/L isopropylthiogalactoside, before recombinant expressed bacterium induced, back cellular lysate thing detects through SDS-PAGE together, electrophoresis detection result as shown in Figure 2, after the SDS-PAGE electrophorogram contrast of recombinant expressed bacterium before inducing, find that recombinant expressed bacterium induces the back a new band of expression (arrow indication band among the figure) to occur at the 42kDa place, the molecular weight size of this band conforms to theoretical derived value, illustrates that the abduction delivering product is the GST-Leptin recombinant protein.
The related technology such as glue recovery, double digestion digestion, carrier connection, intestinal bacteria conversion and IPTG abduction delivering of present embodiment all adopt those skilled in the art's routine operation.
With the e. coli bl21 that contains Rousettus leschenaulti leptin dna recombinant expression plasmid positive colony for preparing among the embodiment 2, by 1: 100 (bacterium liquid: LB
+Substratum, volume ratio) is inoculated in the LB that contains penbritin
+Substratum (yeast extract 5g, peptone 10g, agar 15g, NaCl 5g, ddH
2O to 1 liter, pH=7.4, ammonia benzyl microbiotic 1%) in, 37 ℃, 190rpm cultivates about 3h.Work as OD
600=0.6～1 o'clock, adding IPTG was 0.6mM to working concentration, and in 28 ℃, 190rpm induces 3h.
With the bacterium liquid after above-mentioned the inducing in 5000g, 4 ℃ of centrifugal 10min, thalline is washed 2 times with PBS, during 5000g, 4 ℃ of centrifugal 10min; Add 28ml lysate, 50 μ l N,O-Diacetylmuramidases, 30～40 μ l phenylmethylsulfonyl fluoride (PMSF) (final concentration 100 μ M) to cellular lysate 0.5～1h according to per 2～5ml bacterium liquid; The good thalline of cracking in 4 ℃ or ice bath fragmentation (working hour 3s, interval 5s, 300W) 150 times, about 20～25min, centrifugal (12000g 10min) collects supernatants in 4 ℃ then; After supernatant is considered membrane filtration with 0.45 μ m, as the last sample of next step GST affinity purification.
The filler of the GST affinity purification of present embodiment adopts High-Affinity GST Resin (commercially available), its purifying concrete operations can be with reference to the related reagent specification sheets: filler 4ml, earlier wash post with 25～30ml PBS, add the aforementioned last sample that has prepared then and cross post, flow rate control 2～4s/ drips; PBS with 20 times of column volumes cleans purification column again; The reduced glutathion wash-out: fresh preparation 0.154g gsh+50mlTris-HCl (50mM, PH8.0), each 3ml, flow velocity 4～5s/ drips; Clean purification column 3 times with PBS at last.
The above-mentioned post sample of crossing is carried out ultrafiltration and concentration, can adopt 5000rpm, 4 ℃ of centrifugal 30min.
Add 10U zymoplasm ratio according to every 1mg albumen, adopt zymoplasm (commercially available) that above-mentioned concentrating sample is carried out the normal temperature enzyme and cut 3h, product after enzyme is cut carries out affinity purification once more to remove GST (concrete operations are with aforesaid GST affinity purification), reclaim the post sample and carried out ultrafiltration and concentration (operation is the same) once more, thereby finally obtained Rousettus leschenaulti leptin.With this concentrated supernatant, carrying out sample that the zymoplasm enzyme cuts carries out SDS-PAGE together and detects, detected result as shown in Figure 3, as can be seen from Figure 3, the sample of crossing after column purification and process zymoplasm enzyme are cut obtains two bands, article one, in the 26KD position, according to current material as can be known this band be GST (molecular weight of GST is 26KD), an other band is in the position of 16KD, (the mature peptide sequence that leptin protein of the present invention does not contain signal peptide is 144 amino acid for this and theoretical value, and 144 amino acid proteins encoded are about 15.8KD) conform to, illustrate that Rousettus leschenaulti leptin successfully separates with the GST label, the protein concentration detected result also shows separation simultaneously, the Rousettus leschenaulti leptin concentration and the purity that reclaim reach the cell experiment requirement.
The preceding adipocyte of 3T3-L1 is cultivated in the operation that present embodiment at first adopts conventional cell to cultivate, then with adipocyte before the cultured 3T3-L1 as laboratory sample, the Rousettus leschenaulti leptin (15.8KD) that 3 separation and purification go out to embodiment carries out that MTT detects and the LDH detection.
The MTT of present embodiment detects and LDH detects, and all adopts those skilled in the art's routine operation, with Rousettus leschenaulti leptin (10
-6M) hatch the preceding adipocyte 24h of 3T3-L1, each detects and all establishes two parallel laboratory tests contrasts simultaneously, and one is the blank group of adipocyte before not containing 3T3-L1, and one is employing GST (10
-6M) hatch the control group of adipocyte 24h before the 3T3-L1.
The cytoactive analytical results:
1. Fig. 4 provides the active detected result of Rousettus leschenaulti leptin MTT, and as can be seen from the figure, the data of blank group are 0, with brown flying fox leptin gene recombinant protein (10
-6M) hatch before the 3T3-L1 behind the adipocyte 24h, viable cell ratio (MTT) descends 16.8%, compares difference extremely significantly (P＜0.01) with GST control group (6.6%);
2. Fig. 5 provides the active detected result of Rousettus leschenaulti leptin LDH, and as can be seen from the figure, the data of blank group are 0, with brown flying fox leptin gene recombinant protein (10
-6M) hatch before the 3T3-L1 behind the adipocyte 24h, the serum lactic dehydrogenase fractional release (LDH) of adipocyte significantly raises (4.5%), apparently higher than GST contrast (0.2%) (P＜0.01).
According to the comparing result of Fig. 4 and Fig. 5, illustrate that Rousettus leschenaulti leptin of the present invention has tangible fat acid decomposition activity.
Rousettus leschenaulti leptin and cDNA sequence nucleotide sequence table .txt thereof
SEQUENCE?LISTING
＜110〉Guangdong Province Insects Research Institute
＜120〉Rousettus leschenaulti leptin and cDNA sequence thereof
<130>
<160>7
<170>PatentIn?version?3.5
<210>1
<211>498
<212>DNA
＜213〉brown flying fox (Rousettus leschenaultii)
<400>1
atgcattgtg?gactcctgtg?ccgattcctg?tggctttggc?cttttctctt?ctacattgaa 60
gctgtgccca?tccaaaaagt?ccaggatgac?tccaaaattc?tcatcaagac?gatcatcacc 120
aggatcaata?atattccaca?catgcggtcg?gtctcctcta?aacagacggt?cactggtttg 180
gacttcattc?ctcggctcca?cactgatctg?actttctcca?agatgaacca?gacattagca 240
gtctaccaac?agatccttgc?cgctctgtct?tccagaaatg?tggtccaaat?atccaacgac 300
ctacagaacc?tccaggacct?tctccatctg?ctggccctca?gaagctgtcc?cttcctccag 360
gacagccgcc?cgaatacctt?ggagggcctg?ggtggcatcc?tagaaacctc?attctccatg 420
gaggtggtga?ccctgagcag?gctgcagggg?tttctgcagg?acatgttaca?gcagctggac 480
ctcagccctg?gatgctga 498
<210>2
<211>498
<212>DNA
＜213〉brown flying fox (Rousettus leschenaultii)
<220>
<221>CDS
<222>(1)..(495)
<400>2
atg?cat?tgt?gga?ctc?ctg?tgc?cga?ttc?ctg?tgg?ctt?tgg?cct?ttt?ctc 48
Met?His?Cys?Gly?Leu?Leu?Cys?Arg?Phe?Leu?Trp?Leu?Trp?Pro?Phe?Leu
1 5 10 15
ttc?tac?att?gaa?gct?gtg?ccc?atc?caa?aaa?gtc?cag?gat?gac?tcc?aaa 96
Phe?Tyr?Ile?Glu?Ala?Val?Pro?Ile?Gln?Lys?Val?Gln?Asp?Asp?Ser?Lys
20 25 30
att?ctc?atc?aag?acg?atc?atc?acc?agg?atc?aat?aat?att?cca?cac?atg 144
Ile?Leu?Ile?Lys?Thr?Ile?Ile?Thr?Arg?Ile?Asn?Asn?Ile?Pro?His?Met
35 40 45
cgg?tcg?gtc?tcc?tct?aaa?cag?acg?gtc?act?ggt?ttg?gac?ttc?att?cct 192
Arg?Ser?Val?Ser?Ser?Lys?Gln?Thr?Val?Thr?Gly?Leu?Asp?Phe?Ile?Pro
50 55 60
cgg?ctc?cac?act?gat?ctg?act?ttc?tcc?aag?atg?aac?cag?aca?tta?gca 240
Arg?Leu?His?Thr?Asp?Leu?Thr?Phe?Ser?Lys?Met?Asn?Gln?Thr?Leu?Ala
65 70 75 80
gtc?tac?caa?cag?atc?ctt?gcc?gct?ctg?tct?tcc?aga?aat?gtg?gtc?caa 288
Val?Tyr?Gln?Gln?Ile?Leu?Ala?Ala?Leu?Ser?Ser?Arg?Asn?Val?Val?Gln
85 90 95
ata?tcc?aac?gac?cta?cag?aac?ctc?cag?gac?ctt?ctc?cat?ctg?ctg?gcc 336
Ile?Ser?Asn?Asp?Leu?Gln?Asn?Leu?Gln?Asp?Leu?Leu?His?Leu?Leu?Ala
100 105 110
Rousettus leschenaulti leptin and cDNA sequence nucleotide sequence table .txt thereof
ctc?aga?agc?tgt?ccc?ttc?ctc?cag?gac?agc?cgc?ccg?aat?acc?ttg?gag 384
Leu?Arg?Ser?Cys?Pro?Phe?Leu?Gln?Asp?Ser?Arg?Pro?Asn?Thr?Leu?Glu
115 120 125
ggc?ctg?ggt?ggc?atc?cta?gaa?acc?tca?ttc?tcc?atg?gag?gtg?gtg?acc 432
Gly?Leu?Gly?Gly?Ile?Leu?Glu?Thr?Ser?Phe?Ser?Met?Glu?Val?Val?Thr
130 135 140
ctg?agc?agg?ctg?cag?ggg?ttt?ctg?cag?gac?atg?tta?cag?cag?ctg?gac 480
Leu?Ser?Arg?Leu?Gln?Gly?Phe?Leu?Gln?Asp?Met?Leu?Gln?Gln?Leu?Asp
145 150 155 160
ctc?agc?cct?gga?tgc?tga 498
Leu?Ser?Pro?Gly?Cys
165
<210>3
<211>165
<212>PRT
＜213〉brown flying fox (Rousettus leschenaultii)
<400>3
Met?His?Cys?Gly?Leu?Leu?Cys?Arg?Phe?Leu?Trp?Leu?Trp?Pro?Phe?Leu
1 5 10 15
Phe?Tyr?Ile?Glu?Ala?Val?Pro?Ile?Gln?Lys?Val?Gln?Asp?Asp?Ser?Lys
20 25 30
Ile?Leu?Ile?Lys?Thr?Ile?Ile?Thr?Arg?Ile?Asn?Asn?Ile?Pro?His?Met
35 40 45
Arg?Ser?Val?Ser?Ser?Lys?Gln?Thr?Val?Thr?Gly?Leu?Asp?Phe?Ile?Pro
50 55 60
Arg?Leu?His?Thr?Asp?Leu?Thr?Phe?Ser?Lys?Met?Asn?Gln?Thr?Leu?Ala
65 70 75 80
Val?Tyr?Gln?Gln?Ile?Leu?Ala?Ala?Leu?Ser?Ser?Arg?Asn?Val?Val?Gln
85 90 95
Ile?Ser?Asn?Asp?Leu?Gln?Asn?Leu?Gln?Asp?Leu?Leu?His?Leu?Leu?Ala
100 105 110
Leu?Arg?Ser?Cys?Pro?Phe?Leu?Gln?Asp?Ser?Arg?Pro?Asn?Thr?Leu?Glu
115 120 125
Gly?Leu?Gly?Gly?Ile?Leu?Glu?Thr?Ser?Phe?Ser?Met?Glu?Val?Val?Thr
130 135 140
Leu?Ser?Arg?Leu?Gln?Gly?Phe?Leu?Gln?Asp?Met?Leu?Gln?Gln?Leu?Asp
145 150 155 160
Leu?Ser?Pro?Gly?Cys
165
<210>4
<211>27
<212>DNA
＜213〉artificial sequence
<400>4
Rousettus leschenaulti leptin and cDNA sequence nucleotide sequence table .txt thereof
aagaagmmsa?tccyrggrag?gaaaatg 27
<210>5
<211>25
<212>DNA
＜213〉artificial sequence
<400>5
wgrccttyra?rrcytcagca?yycag 25
<210>6
<211>35
<212>DNA
＜213〉artificial sequence
<400>6
ccggatccgt?gcccatccaa?aaagtccagg?atgac 35
<210>7
<211>25
<212>DNA
＜213〉artificial sequence
<400>7
ccgtcgactc?agcatccagg?gctga 25
Claims (5)
1. leptin protein, the aminoacid sequence of this leptin protein is shown in SEQ ID NO:3.
2. leptin protein according to claim 1 is characterized in that preceding 21 amino acid in this protein amino acid sequence are signal peptide sequence.
3. the cDNA sequence of the leptin protein of encoding, the nucleotide sequence that it is characterized in that this cDNA is shown in SEQ ID NO:1.
4. plasmid that contains the described cDNA sequence of claim 3.
5. a transformant that contains the described plasmid of claim 4 is characterized in that described transformant is an e. coli bl21.
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| CN104829708B (en) * | 2015-05-06 | 2017-11-28 | 广东省生物资源应用研究所 | The leptin activity peptide of one D spiral region mutation and its encoding gene and application |
| CN104829705B (en) * | 2015-05-06 | 2017-11-14 | 广东省生物资源应用研究所 | The leptin activity peptide of one C spiral region mutation and its encoding gene and application |
| CN104829707B (en) * | 2015-05-06 | 2017-12-19 | 广东省生物资源应用研究所 | The leptin activity peptide and its encoding gene and application of one CD ring and E spiral region mutations |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113175A (en) * | 2007-04-28 | 2008-01-30 | 中国科学院西北高原生物研究所 | Rat-rabbit family thin element protein and its cDNA sequence |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113175A (en) * | 2007-04-28 | 2008-01-30 | 中国科学院西北高原生物研究所 | Rat-rabbit family thin element protein and its cDNA sequence |
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| Title |
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| C.Doyon,G.Drouin,V.L.Trudeau,and T.W.Moon.Molecular Evolution of Leptin.《General and Comparative Endocrinology》.2001,第124卷(第2期),第188-198页. * |
| Zhao J,Kunz TH,Tumba N,Schulz LC,Li C,Reeves M,Widmaier EP.Comparative analysis of expression and secretion of placental leptin in mammals.《Am J Physiol Regul Integr Comp Physiol》.2003,第285卷(第2期),第438-446页. * |
| 刘雪梅，刘建宇，魏兰兰，肖鹏，张凤民，谷鸿喜.人类肥胖基因瘦素蛋白的原核表达.《中国微生态学杂志》.2003,第15卷(第2期),第91-92页. * |
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