CN102127163A - Cloning, protein expression, polyclonal antibody preparation and use of nuclear factor TDRP1 - Google Patents
Cloning, protein expression, polyclonal antibody preparation and use of nuclear factor TDRP1 Download PDFInfo
- Publication number
- CN102127163A CN102127163A CN2010100230452A CN201010023045A CN102127163A CN 102127163 A CN102127163 A CN 102127163A CN 2010100230452 A CN2010100230452 A CN 2010100230452A CN 201010023045 A CN201010023045 A CN 201010023045A CN 102127163 A CN102127163 A CN 102127163A
- Authority
- CN
- China
- Prior art keywords
- tdrp1
- expression
- gene
- polyclonal antibody
- nuclear factor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of biology and particularly relates to the expression, polyclonal antibody preparation and use of a novel nuclear factor TDRP1. The amino acid sequence of the novel nuclear factor TDRP1 is represented by SEQ ID NO.2 and the nucleotide coding sequence of the novel nuclear factor TDRP1 is represented by SEQ ID NO.1. The invention also provides the polyclonal antibody of the novel nuclear factor TDRP1. The polyclonal antibody has high specificity and high titer. The TDRP1 is mainly expressed in spermatocytes and can be expressed in cell nuclei. Researches on mats of different ages show that the expression of the Tdrp1 gene and the protein in the testicle tissues of newborn mats is very low, but with the sexual development of the mats, the expression TDRP1 in the cell nuclei grows more and more. The result of the researches indicates that the TDRP1 is an important nuclear factor for the generation of sperms and testicle development.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the Research on Functions of gene clone, genetic expression, protein purification, polyclonal antibody preparation and the TDRP1 of novel nuclear factor TDRP1.
Background technology
Reproduction and growth are the bases that the organism species are continued, and testis is the reproductive organ of buck, are that male sex hormone and sperm produce and sophisticated place.Sperm in testis generation and to reach maturity be the basis that the species genetic material is continued, also be the important content of reproduction.The generation of sperm and maturation are extremely complicated processes, through mitotic division and twice reduction division, a spermatogonium finally develops into four haploid spermatids, spermatid goes through that acrosome forms, nuclear concentrates, flagellum is grown and the process such as lose of kytoplasm, becomes The mature sperm at last.Spermatogeny and maturation are processes that is subjected to strict regulation and control, different times can be expressed different genes and albumen at different androgones, for example: PH-30 and PH-20 gene are expressed [1] [2] at all androgones, 6-phosphomannose acceptor (M6pr) gene that positively charged ion relies on mainly is expressed in spermatocyte and spermatid [3], and fibrous sheath albumen 1 (Fsc1), laminin receptor (Mlr) and cytochrome C s (Cytc
s) wait gene only to express [4-6] at spermatid.
In recent years, large quantities of new genes relevant with testicualr development or spermatogeny are cloned successively, in this respect, the researcher of China has been made unremitting effort, they have cloned the new genes relevant with testicualr development or spermatogeny such as LM23, TSARG2, MSRG-11, NHEDC1, NYD-SP15, Pkd212, SPATA12, TSAP, Tsc24, TSC77, Ube1, CKLFSF2, AEP1, Znf313, and preliminary study has been studied their function [7-20].Some external research groups, applying gene is rejected animal model, has deeply studied some gene relevant with testicualr development or spermatogeny clearly.Discover that the male mice of gene knockouts such as CIB1, Ant4, PATZ1, Sirt1, cyclin A1, Hsp70-2, Trf2 and Siahla has all occurred sterile, what have also dies with the accent of sexual cell and the dwindling of testis [21-28].
Relevant references:
1、Wolfsberg?TG，Bazan?JF，Blobel?CP，Myles?DG，Primakoff?P，White?JM.Theprecursor?region?of?a?protein?active?in?sperm-egg?fusion?contains?a?metalloproteaseand?a?disintegrin?domain：structural，functional，and?evolutionary?implications.ProcNatl?Acad?Sci?U?S?A.1993Nov?15；90(22)：10783-7.
2、Lin?Y，Mahan?K，Lathrop?WF，Myles?DG，Primakoff?P.A?hyaluronidase?activity?ofthe?sperm?plasma?membrane?protein?PH-20?enables?sperm?to?penetrate?the?cumuluscell?layer?surrounding?the?egg.J?Cell?Biol.1994?Jun；125(5)：1157-63.
3、O′Brien?DA，Welch?JE，Fulcher?KD，Eddy?EM.Expression?of?mannose?6-phosphatereceptor?messenger?ribonucleic?acids?in?mouse?spermatogenic?and?Sertoli?cells.BiolReprod.1994Feb；50(2)：429-35.
4、Fulcher?KD，Mori?C，Welch?JE，O′Brien?DA，Klapper?DG，Eddy?EM.Characterization?of?Fsc1?cDNA?for?a?mouse?sperm?fibrous?sheath?component.BiolReprod.1995Jan；52(1)：41-9.
5、Fulcher?KD，Welch?JE，Davis?CM，O′Brien?DA，Eddy?EM.Characterization?oflaminin?receptor?messenger?ribonucleic?acid?and?protein?expression?in?mousespermatogenic?cells.Biol?Reprod.1993Mar；48(3)：674-82.
6、Hake?LE，Hecht?NB.Utilization?of?an?alternative?transcription?initiation?site?ofsomatic?cytochrome?c?in?the?mouse?produces?a?testis-specific?cytochrome?c?mRNA.JBiol?Chem.1993?Mar?5；268(7)：4788-97.
7、Liu?ML，Pei?KY，Shi?XQ，Liu?DY，Jia?MC.Identification?and?characterization?of?anovel?spermatogenesis?related?gene?LM23?in?rat?testis.Biochem?Biophys?ResCommun.2007May?11；356(3)：576-81.
8、Liu?SF，Lu?GX，Liu?G，Xing?XW，Li?LY，Wang?Z(2004)Cloning?of?a?full-lengthcDNA?of?human?testis-specific?spermatogenic?cell?apoptosis?inhibitor?TSARG2as?acandidate?oncogene.Biochem?Biophys?Res?Commun?319，32-40.
9、Deng?Y，Nie?DS，Wang?J，Tan?XJ，Nie?ZY，Yang?HM，Hu?LS，Lu?GX(2005)Molecularcloning?of?MSRG-11?gene?related?to?apoptosis?of?mouse?spermatogeniccells.Acta?Biochim?Biophys?Sin?37，159-166.
10、Ye?G，Chen?C，Han?D，Xiong?X，Kong?Y，Wan?B，Yu?L?Cloning?of?a?novelhuman?NHEDC1(Na+/H+exchanger?like?domain?containing?1)gene?expressedspecifically?in?testis.Mol?Biol?Rep.2006?Sep；33(3)：175-80.
11、Liu?Q，Liu?J，Cao?Q，Sha?J，Zhou?Z，Wang?H，Li?J.NYD-SP15：a?novel?genepotentially?involved?in?regulating?testicular?development?and?spermatogenesis.Biochem?Genet.2006?Oct；44(7-8)：409-23.
12、Chen?Y，Zhang?Z，Lv?XY，Wang?YD，Hu?ZG，Sun?H，Tan?RZ，Liu?YH，Bian?GH，Xiao?Y，Li?QW，Yang?QT，Ai?JZ，Feng?L，Yang?Y，Wei?YQ，Zhou?Q.Expression?ofPkd212?in?testis?is?implicated?in?spermatogenesis.Biol?Pharm?Bull.2008Aug；31(8)：1496-500.
13、Dan?L，Lifang?Y，Guangxiu?L.Expression?and?possible?functions?of?a?novel?geneSPATA12?in?human?testis.J?Androl.2007?Jul-Aug；28(4)：502-12.
14、Bin?L，Gang?W，Hu?J，Gong?W，Yue?M，Song?P.Identification?andcharacterization?of?TSAP，a?novel?gene?specifically?expressed?in?testis?duringspermatogenesis.Mol?Reprod?Dev.2007Sep；74(9)：1141-8.
15、Tang?A，Yu?Z，Gui?Y，Guo?X，Long?Y，Cai?Z.Identification?and?characteristics?ofa?novel?testis-specific?gene，Tsc24，in?human?and?mice.Biol?Pharm?Bull.2006Nov；29(11)：2187-91.
16、Tang?A，Yu?Z，Gui?Y，Zhu?H，Long?Y，Cai?Z.Identification?of?a?noveltestis-specific?gene?in?mice?and?its?potential?roles?in?spermatogenesis.Croat?Med?J.2007Feb；48(1)：43-50.
17、DU?Y，Liu ML，Jia?MC.Identification?and?characterization?of?aspermatogenesis-related?gene?Ubel?in?rat?testis.Sheng?Li?Xue?Bao.2008?Jun25；60(3)：382-90.
18、Liu?G，Xin?ZC，Chen?L，Tian?L，Yuan?YM，Song?WD，Jiang?XJ，Guo?YL.Expression?and?localization?of?CKLFSF2?in?human?spermatogenesis.Asian?J?Androl.2007?Mar；9(2)：189-98.
19、Luk?JM，Lee?NP，Shum?CK，Lam?BY，Siu?AF，Che?CM，Tam?PC，Cheung?AN，Yang?ZM，Lin?YN，Matzuk?MM，Lee?KF，Yeung?WS.Acrosome-specific?gene?AEP1：identification，characterization?and?roles?in?spermatogenesis.J?Cell?Physiol.2006Dec；209(3)：755-66.
20、Li?N，Sun?H，Wu?Q，Tao?D，Zhang?S，Ma?Y.Cloning?and?expression?analysis?of?anovel?mouse?zinc?finger?protein?gene?Znf313?abundantly?expressed?in?testis.JBiochem?Mol?Biol.2007?Mar?31；40(2)：270-6.
21、Yuan?W，Leisner?TM，McFadden?AW，Clark?S，Hiller?S，Maeda?N，O′Brien?DA，Parise?LV.CIB1?is?essential?for?mouse?spermatogenesis.Mol?Cell?Biol.2006Nov；26(22)：8507-14.
22、Brower?JV，Rodic?N，Seki?T，Jorgensen?M，Fliess?N，Yachnis?AT，McCarrey?JR，Oh?SP，Terada?N.Evolutionarily?conserved?mammalian?adenine?nucleotidetranslocase?4?is?essential?for?spermatogenesis.J?Biol?Chem.2007?Oct5；282(40)：29658-66.
23、Fedele?M，Franco?R，Salvatore?G，Paronetto?MP，Barbagallo?F，Pero?R，ChiariottiL，Sette?C，Tramontano?D，Chieffi?G，Fusco?A，Chieffi?P.PATZ1?gene?has?a?criticalrole?in?the?spermatogenesis?and?testicular?tumours.J?Pathol.2008?May；215(1)：39-47.
24、Coussens?M，Maresh?JG，Yanagimachi?R，Maeda?G，Allsopp?R.Sirtl?deficiencyattenuates?spermatogenesis?and?germ?cell?function.PLoS?ONE.2008?Feb13；3(2)：e1571.
25、Liu?D，Matzuk?MM，Sung?WK，Guo?Q，Wang?P&?Wolgemuth?DJ?1998?CyclinA1?is?required?for?meiosis?in?the?male?mouse.Nature?Genetics?20377-380.
26、Dix?DJ，Allen?JW，Collins?BW，Mori?C，Nakamura?N，Poorman-Allen?P，Goulding?EH?&?Eddy?EM?1996?Targeted?gene?disruption?of?Hsp70-2?results?in?failedmeiosis，germ?cell?apoptosis，and?male?infertility.Proceedings?of?the?NationalAcademy?of?Sciences?of?the?United?States?of?America?93?3264-3268.
27、Zhang?D，Penttila?TL，Morris?PL，Teichmann?M?&?Roeder?RG?2001Spermiogenesis?deficiency?in?mice?lacking?the?Trf2?gene.Science?292?1153-1155.
28、Dickins?RA，Frew?IJ，House?CM，O’Bryan?MK，Holloway?AJ，Haviv?I，TraficanteN，de?Kretser?DM?&?Bowtell?DD?2002?The?ubiquitin?ligase?component?Siahla?isrequired?for?completion?of?meiosis?I?in?male?mice.Molecular?and?Cellular?Biology?222294-2303.
Summary of the invention
The purpose of this invention is to provide a novel nuclear factor TDRP1.
Another object of the present invention provides the preparation method of novel nuclear factor TDRP1.
The purpose of this invention is to provide the application of novel nuclear factor TDRP1.
The present invention is according to information biology and molecular biological analytical results, and using gene engineering and protein engineering are carried out protein expression and purifying and Polyclonal Antibody Preparation.Comprise the protein of expressing total length TDRP1 encoding sequence, the clone comprises expression total length TDRP1 encoding sequence in suitable expression vector, with this carrier transfection host cell, cultivate this host cell under the condition of this dna segment being suitable for expressing, and from culture, reclaim and the required TDRP1 albumen of purifying.
2004, gene expression profile (Wang XC, et al.Endocr-Relat Cancer, 2004 of insulin human's tumor tissue were disclosed; 11 (2): 295-303.).A new gene is cloned in the present invention from insulin human's tumor tissue, with its called after testicualr development associated protein (
TEstis
DEvelopment
RElated
PRotein, TDRP).Human TDRP gene chromosomal localization is in 8p23, and size is 55.03Kb, and two different transcripts are arranged, and size is respectively 3.2Kb and 2.4Kb.3.2Kb transcript (TDRP1) 3 exons are arranged, encoding sequence comprises 549bp, the coding 183 amino-acid residues albumen.2.4Kb transcript (TDRP2) be that No. 3 exon by the 3.2Kb transcript cuts off 788 Nucleotide and produce, it comprises 4 exons, reading frame has 594 Nucleotide, 198 the amino acid whose albumen of encoding fully.163 amino acid of their 5 ' ends of TDRP1 and TDRP2 albumen are identical.There are this two transcripts in the testis tissue that Northern blotting and RT-PCR detect the people, and finds that TDRP1 is the main transcript of TDRP gene, so the focusing on the TDRP1 transcript of this research.PCR product subclone is to the pDrive carrier, and order-checking has confirmed the nucleotide sequence of two transcripts.Use NetNES 1.1 softwares (http://www.cbs.dtu.dk/services/NetNES/) and find that the TDRP intramolecularly contains the abundant nuclear output sequence of tangible leucine.
With the SD rat is animal model, uses the distribution expression pattern that Northern blotting has set up rat Tdrp gene, find that the Tdrp gene mainly is expressed in testis tissue, and expression amount is extremely abundant, at kidney and fatty tissue a spot of expression is arranged also.The result finds that also also there are two different transcripts in rat Tdrp gene, and is similar with the 2.4Kb transcript with the 3.2Kb of people TDRP gene, and bigger transcript (Tdrp1) is main transcript.Use prokaryotic expression carrier: the pGEX-4T-2 expression and purification GST-TDRP1 fusion rotein, obtained the polyclonal antibody of TDRP1 behind the immunizing rabbit.The immunohistochemical methods method has been studied the cellular localization of TDRP1 in rat testis, finds that TDRP1 is expressed in androgone, and specifically, TDRP1 mainly is expressed in spermatocyte, at spermatogonium and spermatid a spot of expression is arranged also.And do not have expression at bad enlightening cell (Leydigcell) and sertoli cell (Sertoli cell).Find that simultaneously TDRP1 can be expressed in the nucleus of some androgone.Research to some cell strain of deriving from the mouse testis tissue has also confirmed The above results, Tdrp1 gene and albumen have expression in spermatogonium strain GC-1 spg and spermatocyte strain GC-2 spd, and do not have expression at bad enlightening cell strain TM3 and sertoli cell strain TM4.The encoding sequence of TDRP1 gene is cloned in the pEGFP-C2 carrier, and transfection HeLa cell, laser co-focusing discovers that the GFP-TDRP1 fusion rotein is distributed in endochylema and karyon; Immunofluorescence research simultaneously finds that also endogenic TDRP1 has expression at endochylema and karyon in the HeLa cell.Respectively behind extracting endochylema and the karyon albumen, Western blotting discovers, no matter in transfection the HeLa nucleus of TDRP1 gene, or the nucleus extract of adult rat testis tissue all can detect the expression of TDRP1.Above-mentioned research prompting, TDRP1 is a novel nf at the testis spermatogenic cell expressed in abundance.
To different developmental age rat discover that Tdrp1 gene and albumen are extremely low at new life's rat testis expression amount, along with the increase at age, expression amount rises, peak in back 2 months (sexual development maturation) times of birth, aging along with the age later on, expression amount slightly descends.The effect of prompting TDRP1 may be relevant with testicualr development or spermatogeny.In addition, the research of protein level also finds, along with the maturation of rat sexual development, the expression of TDRP1 in nucleus is more and more, and the going into to check in testicualr development or spermatogeny of prompting TDRP1 has vital role.The using yeast two-hybrid system, screen 7 can with the interactional albumen of TDRP1, RANBP9 be one of them can with the interactional albumen of TDRP1, this effect is by experiment confirms such as GST pull down and co-immunoprecipitations, because existing studies confirm that, RANBP9 can interact with androgen receptor, activates the transcriptional activity of androgen receptor.Therefore, we infer that TDRP1 by with after RANBP9 combines, regulates the transcriptional activity of androgen receptor, thereby plays a significant role in spermatogeny or testicualr development process.
The invention provides a kind of nf TDRP1, its aminoacid sequence is shown in SEQ ID NO 2.Its nucleotide sequence is shown in SEQ ID NO 1.SEQ ID NO 2 (aminoacid sequence of TDRP1): MWKLGRGRVLLDEPPEEEDGLRGGPPPAAAAQAQVQGASFRGWKEVTSLFNKDDEQ HLLERCKSPKSKGTNLRLKEELKAEKKSGFWDNLVLKQNIQSKKPDEIEGWEPPKL ALEDISADPEDTVGGHPSWSGWEDDAKGSTKYTSLASSANSSRWSLRAAGRLVSIR RQSKGHLTDSPEEAE.
Nf TDRP1 of the present invention also comprises having variant form identical functional transcription factor, SEQ ID NO:2.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.
The polynucleotide sequence of coding nf TDRP1 is selected from the 81-629 position among the sequence table SEQ ID NO:1.In the present invention, the polynucleotide encoding sequence of nf TDRP1 also comprises degenerate sequence.Degenerate sequence is meant, having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, so be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out with 81-629 position nucleotide sequence homology among the SEQ ID NO:1.
The preparation method of described nf TDRP1 can be according to the aminoacid sequence synthetic of SEQ ID NO 2.Also can utilize ordinary method, with nf TDRP1 separation and purification from people's cell or tissue (for example insulin human's tumor tissue).The polynucleotide sequence of nf TDRP1 of perhaps will encoding is cloned into expression vector, changes host cell expression over to, and final separation and purification obtains nf TDRP1 albumen.For example, adopt prokaryotic vector pGEX-4T-2, the host can be ROSSET (DE3) or BL21 (DE3).
Among the present invention, the preparation of TDRP1 gene comprises after the PCR reaction carries out purifying, and whether nucleotide sequence analysis checking gene is correct.The amplification of total length TDRP1 gene coded sequence of the present invention does not limit specific archaeal dna polymerase.In a preferred embodiment, the present invention uses the Advantage GC2PCR test kit of CLONTECH company.
The present invention also provides the cloning vector or the expression vector of the nucleotide coding sequence that contains the TDRP1 gene.Usually the nucleotide coding sequence of TDRP1 gene can be inserted the multiple clone site of commercially available cloning vector or expression vector and get.For example, adopt prokaryotic vector pGEX-4T-2.
The present invention also provides the polyclonal antibody of above-mentioned nf TDRP1.This polyclonal antibody can obtain according to the ordinary method of this area, also can prepare according to following concrete grammar:
(1) obtains the complete encoding sequence gene;
(2) with gene and prokaryotic expression carrier reorganization;
(3) recombinant expression vector is transduceed prokaryotic expression system;
(4) induce the expression of TDRP1 with 1mM IPTG, the TDRP1 after the expression is through gsh affinity chromatography purifying in addition, and excises the GST label with zymoplasm from fusion rotein and obtain the good TDRP1 albumen of purifying;
(5) immunity of the TDRP1 behind purifying New Zealand white rabbit obtains TDRP1 polyclonal antibody serum.
Can utilize Western blotting method to identify the quality and the titre of TDRP1 polyclonal antibody.
DNA and expression vector reorganization with total length TDRP1 encoding sequence of the present invention form recombinant expression plasmid.The present invention does not limit specific expression plasmid.In a preferred embodiment, the present invention uses prokaryotic expression carrier pGEX-4T-2.
Simultaneously above-mentioned recombinant prokaryotic expression vector clone is imported suitable prokaryotic host cell according to a conventional method.It the invention is not restricted to specific host cell, as long as can express described recombinant expression vector.Described prokaryotic expression system can be ROSSET (DE3) or BL21 (DE3).In a preferred version, the present invention uses intestinal bacteria ROSSET (DE3) etc.
Expression product of the present invention is present in the cell space of host cell with the form of inclusion body, crack the separation inclusion body, high concentration urea dissolving inclusion body, separation and purification TDRP1, albumen behind the purifying is carried out the SDS-PAGE electrophoresis, gel is put into after the dyeing of Coomassie brilliant blue dye liquor, the decolouring, cuts off with the aseptic operation blade and contains the proteic purpose band of TDRP1.
The present invention adopts new zealand white rabbit as immune animal.To contain the proteic gel of TDRP1 and pulverize, with Freund's complete adjuvant emulsification, multiple injection immune rabbit, immune serum is isolated in the heart blood sampling after the last immunity, adopts Western Blot method to detect the quality and the titre of antibody.
The TDRP1 polyclonal antibody that utilization of the present invention prepares adopts the immunohistochemical methods method to study the cellular localization of TDRP1 in rat testis, find that TDRP1 mainly is expressed in spermatocyte, at spermatogonium and spermatid a spot of expression is arranged also, and not having expression at bad enlightening cell (Leydig cell) and sertoli cell (Sertolicell), immunohistochemical methods finds that also TDRP1 can express at the nucleus of androgone simultaneously.
The present invention is cloned into the encoding sequence of TDRP1 gene in the pEGFP-C2 carrier, and transfection HeLa cell, and laser co-focusing discovers that the GFP-TDRP1 fusion rotein is distributed in endochylema and karyon; Immunofluorescence research simultaneously finds that also endogenic TDRP1 has expression at endochylema and karyon in the HeLa cell.Respectively behind extracting endochylema and the karyon albumen, Western blotting discovers, no matter in transfection the HeLa nucleus of TDRP1 gene, or the nucleus extract of adult rat testis tissue all can detect the expression of TDRP1.Above-mentioned research prompting, TDRP1 is a novel nf.
Novel nuclear factor TDRP1 plays a significant role in spermatogeny or testicualr development process.
The present invention discovers that to difference developmental age rat Tdrp1 gene and albumen are extremely low at new life's rat testis expression amount, along with the increase at age, expression amount rises, peak in back 2 months (sexual development maturation) times of birth, aging along with the age later on, expression amount slightly descends.The effect of prompting TDRP1 may be relevant with testicualr development or spermatogeny.In addition, the research of protein level also finds, along with the maturation of rat sexual development, the expression of TDRP1 in nucleus is more and more, and the going into to check in testicualr development or spermatogeny of prompting TDRP1 has vital role.
Using yeast two-hybrid system of the present invention, screen 7 can with the interactional albumen of TDRP1, RANBP9 be one of them can with the interactional albumen of TDRP1, this effect is by experiment confirms such as GST pull down and co-immunoprecipitations, because existing studies confirm that, RANBP9 can interact with androgen receptor, activates the transcriptional activity of androgen receptor.Therefore, we infer that TDRP1 by with after RANBP9 combines, can regulate the transcriptional activity of androgen receptor.
All basic molecular biology operative techniquies are all with reference to " the molecular cloning third edition " in the above technical scheme.
Using gene engineering method of the present invention is produced and is obtained good, the efficient and high-quality TDRP1 polyclonal antibody of specificity, for physiological function and the mechanism of action thereof of further inquiring into TDRP1 lays the foundation.Utilize molecular biology method tentatively to disclose the function of TDRP1 simultaneously, prompting TDRP1 plays a significant role in spermatogeny or testicualr development process, for the treatment of following infertility provides new approach.
A novel nuclear factor-TDRP1 has been cloned in the present invention first, expression and purification TDRP1 albumen, prepared the TDRP1 polyclonal antibody.Immunohistochemical methods shows that TDRP1 mainly is expressed in androgone at testis tissue.Laser co-focusing, immunofluorescence and Western blotting etc. test demonstration, and TDRP1 can be expressed in nucleus, and prompting TDRP1 is a nf.To different developmental age rat discover that Tdrp1 gene and albumen are extremely low at new life's rat testis expression amount, to birth 3 weeks of back, expression amount obviously rises, peak in back 2 months (sexual development maturation) times of birth, aging along with the age later on, expression amount slightly descends.In addition, the research of protein level also finds, along with the maturation of rat sexual development, the expression of TDRP1 in nucleus is more and more, and the going into to check in testicualr development or spermatogeny of prompting TDRP1 has vital role.The using yeast two-hybrid system, screen 7 can with the interactional albumen of TDRP1, RANBP9 be one of them can with the interactional albumen of TDRP1, this effect is by experiment confirms such as GST pull down and co-immunoprecipitations, after we infer TDRP1 and RANBP9 combine, regulate the transcriptional activity of androgen receptor, thereby in spermatogeny or testicualr development process, play a significant role, may provide new target spot to the treatment of following infertility the further research of TDRP1 function.
Embodiment
Embodiment 1TDRP1 construction of prokaryotic expression vector
The dna fragmentation and the pGEX-4T-2 that will contain total length TDRP1 gene coded sequence carry out double digestion (BamHI and Sal I), connect with the T4 ligase enzyme, set up the TDRP1 prokaryotic expression carrier.With recombinant vectors pGEX-4T-2-TDRP1 transformed into escherichia coli ROSSET (DE3), ampicillin medium screening positive strain, the plasmid enzyme restriction evaluation is extracted in amplification and order-checking confirms that sequence is correct.
The expression of embodiment 2TDRP1
Choose the mono-clonal colony inoculation to 5ml YTA medium, 37 ℃ * 250rpm to OD600=1-2, be seeded to 500ml YTA medium, 37 ℃ * 250rpm to OD600=1-2, add 1M IPTG to final concentration 1mM, 25 ℃ of abduction delivering 3h, 4 ℃ of 4000rpm 20min, collect thalline, the ultrasonic degradation bacterium is also centrifugal, after will going up cleer and peaceful precipitation (0.01%PBS dissolving) and using the mixing of 5 * sample-loading buffer respectively, make the SDS-PAGE electrophoresis, after the Coomassie brilliant blue dyeing, as seen induce in the postprecipitation band that concentrates at the about 47Kd of molecular weight place, and expression amount is low in the supernatant.Prompting TDRP1 protein expression is in inclusion body.
The proteic purifying of embodiment 3 TDRP1
Full bacterium is centrifugal after will inducing, and with BugBuster (Novagen company) and ultrasonic degradation bacterium, recentrifuge, centrifuged deposit dissolves with the 1 * binging buffer that contains 8M urea, the dissolved supernatant B-PER of Thermo company
TMBacterium GST tag fusion protein pillar purification kit, obtain the good GST-TDRP1 fusion rotein of purifying, from fusion rotein, excise the GST label with zymoplasm, behind the proteolysis, GST carrier and proteolytic enzyme are removed by the glutathione agarose affinity chromatography, obtain the good TDRP1 albumen of purifying at last.
The preparation of embodiment 4TDRP1 polyclonal antibody
The TDRP1 albumen of above-mentioned purifying is carried out the SDS-PAGE electrophoresis, gel is put into the Coomassie brilliant blue dye liquor dyeed about 4 hours, the taking-up gel is put into destainer and was decoloured about 4～8 hours, cuts off required purpose band with the aseptic operation blade, 1 * PBS washing 2 times, 4 ℃ of preservations are standby.Adopt new zealand white rabbit as immune animal.Blood sample collection was to make negative control before white rabbit made immunization.Pulverize containing the proteic gel of 100ugTDRP1 approximately, with Freund's complete adjuvant emulsification, after hair is scraped at the white rabbit back, disperse the 24-36 site injection, behind the initial immunity 4,7,9,11 weeks respectively with the antigen of equivalent and Freund's incomplete adjuvant emulsification after booster immunization.Heart blood sampling after the last immunity detects antibody titer with Western Blot method.Immune serum is isolated in carotid artery bloodletting in the 10th day after the last immunity, and immune serum adds NaN
3To final concentration 0.02%, be sub-packed in the 1.5ml centrifuge tube ,-80 ° of preservations are standby.Adopt Western Blot method to detect antibody titers and quality.
The preliminary announcement of embodiment 5TDRP1 function
(1) cellular localization of TDRP1 albumen in testis tissue
The testis tissue paraffin section of preparation rat, the immunohistochemical methods method has been studied the cellular localization of TDRP1 in rat testis, find that TDRP1 is expressed in androgone, specifically, TDRP1 mainly is expressed in spermatocyte, at spermatogonium and spermatid a spot of expression is arranged also, and do not have expression at bad enlightening cell (Leydig cell) and sertoli cell (Sertoli cell).Find that simultaneously TDRP1 can be expressed in the nucleus of some androgone.
(2) TDRP1 is a novel nf
Use NetNES 1.1 softwares (http://www.cbs.dtu.dk/services/NetNES/) and find that the TDRP intramolecularly contains the abundant nuclear output sequence of tangible leucine.The encoding sequence of TDRP1 gene is cloned in the pEGFP-C2 carrier, and transfection HeLa cell, laser co-focusing discovers that the GFP-TDRP1 fusion rotein is distributed in endochylema and karyon; Immunofluorescence research simultaneously finds that also endogenic TDRP1 has expression at endochylema and karyon in the HeLa cell.Respectively behind extracting endochylema and the karyon albumen, Western blotting discovers, no matter in transfection the HeLa nucleus of TDRP1 gene, or the nucleus extract of adult rat testis tissue all can detect the expression of TDRP1.Above-mentioned research prompting, TDRP1 is a novel nf at the testis spermatogenic cell expressed in abundance.
The preliminary Research on Functions of embodiment 6TDRP1 in spermatogeny or testicualr development process
To different developmental age rat discover that Tdrp1 gene and albumen are extremely low at new life's rat testis expression amount, to birth 3 weeks of back, expression amount obviously rises, peak in back 2 months (sexual development maturation) times of birth, aging along with the age later on, expression amount slightly descends.In addition, the research of protein level also finds, along with the maturation of rat sexual development, the expression of TDRP1 in nucleus is more and more, and the going into to check in testicualr development or spermatogeny of prompting TDRP1 has vital role.The using yeast two-hybrid system, screen 7 can with the interactional albumen of TDRP1, RANBP9 be one of them can with the interactional albumen of TDRP1, this effect is by experiment confirms such as GST pull down and co-immunoprecipitations, because existing studies confirm that, RANBP9 can interact with androgen receptor, activates the transcriptional activity of androgen receptor.Therefore, we infer that TDRP1 by with after RANBP9 combines, regulates the transcriptional activity of androgen receptor, thereby plays a significant role in spermatogeny or testicualr development process.
From the above description, those skilled in the art can understand essential characteristic of the present invention at an easy rate.And under the situation that does not depart from its purport and scope, can carry out various changes and improvement to the present invention.Those skilled in the art can use the present invention to greatest extent.Therefore, above-mentioned preferred specific embodiments should be understood that only to illustrate, but not limits the scope of the invention by any way.
Sequence table
<210>1
<211>3273
<212>DNA
<213>Homo?sapiens
<220>
<221>CDS
<222>(81)..(629)
<223>
<400>1
ggcagagcgt?ccggcggccg?ggagggacgc?ggacccacag?cccgacgcac?ggacggaggg 60
acgccggagc?ccgcctgacc?atg?tgg?aag?ctg?ggc?cgg?ggc?cga?gtg?ctg?ctg 113
Met?Trp?Lys?Leu?Gly?Arg?Gly?Arg?Val?Leu?Leu
1 5 10
gac?gag?ccc?ccc?gag?gag?gag?gac?ggc?ctg?cgt?ggg?ggg?ccg?cca?ccg 161
Asp?Glu?Pro?Pro?Glu?Glu?Glu?Asp?Gly?Leu?Arg?Gly?Gly?Pro?Pro?Pro
15 20 25
gcc?gcc?gcc?gcc?cag?gcg?cag?gtt?cag?gga?gca?agt?ttc?cga?ggt?tgg 209
Ala?Ala?Ala?Ala?Gln?Ala?Gln?Val?Gln?Gly?Ala?Ser?Phe?Arg?Gly?Trp
30 35 40
aaa?gaa?gtg?act?tca?ctg?ttt?aac?aaa?gat?gat?gag?cag?cat?ctc?ctg 257
Lys?Glu?Val?Thr?Ser?Leu?Phe?Asn?Lys?Asp?Asp?Glu?Gln?His?Leu?Leu
45 50 55
gaa?aga?tgt?aaa?tct?ccc?aag?tcc?aaa?gga?act?aac?tta?cga?tta?aaa 305
Glu?Arg?Cys?Lys?Ser?Pro?Lys?Ser?Lys?Gly?Thr?Asn?Leu?Arg?Leu?Lys
60 65 70 75
gaa?gag?ttg?aag?gca?gag?aag?aaa?tct?gga?ttt?tgg?gac?aat?ttg?gtt 353
Glu?Glu?Leu?Lys?Ala?Glu?Lys?Lys?Ser?Gly?Phe?Trp?Asp?Asn?Leu?Val
80 85 90
tta?aaa?cag?aat?ata?cag?tct?aaa?aaa?cca?gat?gaa?att?gaa?ggt?tgg 401
Leu?Lys?Gln?Asn?Ile?Gln?Ser?Lys?Lys?Pro?Asp?Glu?Ile?Glu?Gly?Trp
95 100 105
gag?cct?cca?aaa?ctt?gct?ctt?gaa?gac?ata?tcg?gct?gac?cct?gag?gac 449
Glu?Pro?Pro?Lys?Leu?Ala?Leu?Glu?Asp?Ile?Ser?Ala?Asp?Pro?Glu?Asp
110 115 120
acc?gtg?ggt?ggc?cac?cca?tcc?tgg?tca?ggc?tgg?gag?gat?gac?gcc?aag 497
Thr?Val?Gly?Gly?His?Pro?Ser?Trp?Ser?Gly?Trp?Glu?Asp?Asp?Ala?Lys
125 130 135
ggc?tcg?acc?aag?tac?acc?agc?ctg?gcc?agc?tct?gcc?aac?agc?tcc?agg 545
Gly?Ser?Thr?Lys?Tyr?Thr?Ser?Leu?Ala?Ser?Ser?Ala?Asn?Ser?Ser?Arg
140 145 150 155
tgg?agc?ctg?cgc?gcg?gca?ggg?agg?ctg?gtg?agc?atc?cga?cgg?cag?agt 593
Trp?Ser?Leu?Arg?Ala?Ala?Gly?Arg?Leu?Val?Ser?Ile?Arg?Arg?Gln?Ser
160 165 170
aaa?ggc?cac?ctg?aca?gat?agc?ccg?gag?gag?gcg?gag?tgaggggggc 639
Lys?Gly?His?Leu?Thr?Asp?Ser?Pro?Glu?Glu?Ala?Glu
175 180
tgcgtggcaa?gtgtgccccg?acatggtggc?cttttatgag?tataccatgt?agttgttgag 699
tcttttccgc?gttagaaaga?atagagagtc?tactttttgc?ctatatttga?tatttggacc 759
tctgtttctt?tatttaatga?actctcacac?acactgtgac?tccttggtga?acacgtgcag 819
ggattgtaca?tttgattgcc?ttattgcaga?aatattcacc?tatcagtcca?tgttttgcag 879
aaactggaga?tgtgaattta?tgatgctctc?ccataataca?gcagtaatga?tttgcagttt 939
ctcacaaaat?gattttcatg?ctgctctgtg?ttgtagttct?gtttcagaac?ttccgtacct 999
ttttcttgta?tgtagcacgt?ataaatgcag?ctgtcatgca?gttcttttct?tttgctagaa 1059
aattagtcag?gaggtaagat?gaatcttcca?aagttatgtt?aaattttgtt?taacttgaca 1119
aattaaactt?tgttcttatt?aaacaaatac?gtaaacaaat?actggaaaag?caaagcttat 1179
atttgggagt?aaaatgtatc?ttaaaatgca?tgttcatctt?ttgctgaggg?aggaatagct 1239
tcttgtatgt?ccactggata?gtaacagtgg?ttcttttttg?gaattttatt?tttacctctt 1299
ttgaacttct?aaatcggtcc?ctttattttc?ttaaaacatt?tgctctcttt?tttgtttaca 1359
gattaaagaa?gtatggatca?attttagtca?gttgataatt?tcatttagga?aacattgtct 1419
tgctcattat?agagagttaa?gactgtgcat?aaaatactga?agtgcctctt?ttttattagg 1479
ctcatatgtt?tgttcttgca?aagcgagatg?cctattgtga?ttggtgttag?atctgtaaga 1539
agctgtgttt?tttccacaag?aataatcgac?gaccggtttg?agagcatctc?tcgcgttctt 1599
tttctgtctg?cttactgtcc?ttgacaatgt?gatcctgcac?agtatcaggt?gccagttatg 1659
cgtccctgga?aggttggctc?ttatcagcaa?gtcctgcaga?tatctcttag?ataaaagtgg 1719
tttgaaagaa?aaggtatatt?tttctttttt?gtttaaaagt?aaaataaaac?ctccagttac 1779
actgtgcatt?cccctcacgt?aaaaacaaga?caaaaaccct?ttcctgagtt?gctcggcatg 1839
cactcctgtg?ctgtttcatg?catgtggatg?ttccttccgt?ttgttcctgt?ggaataactg 1899
agtgtgcctg?atggcagaac?acactgcagt?gttatcagtg?tctgcatgtt?ttttaataga 1959
acaggtttac?ttgatctgtc?atctgttatg?gaaaaaacag?caattacttt?tgcatccatc 2019
tagctaaatc?tataatctgt?gtcaatcact?tatacctatc?aatcatccgt?atctattcac 2079
ctgtcctcta?tgtctgtctt?ctctcagtct?acatccatct?agccttctgt?caatcatcta 2139
cttttttttt?taatagaaca?agaagtttac?ttatcaagtc?ttgaaagggg?gacatgtttc 2199
aattggtgtt?caaacaaaaa?ttccagcttg?tattagaacc?ttgaaatggt?gaagttgtgg 2259
aggttttctt?tgttccataa?tacagagaca?agttcataat?ttttagtata?acagctaagt 2319
tgacaaattc?taagtttcct?caggtaaata?gtcgtaactg?tccttttccc?tagagaagtg 2379
cttgctggga?tagtaaaaac?atacaccatt?tcatgacttc?tgcaaatact?ttccagcgga 2439
agtcagtgta?ggtttgtttt?tggctaacat?ggtcttgctg?cgcaggaatg?taaacactgt 2499
gtttgaaact?ctggaaatca?cgtgtgtggg?gagatgggga?cgcttcccat?gttgtgggga 2559
gctctgtggc?tgtgatggct?gcagttgccg?tgcctctgtt?ggaacgccaa?gtgcctgcaa 2619
ctcacgtcaa?tcatagaatt?gtgacgcaca?gttggcaaaa?tagttcttta?tgctatttct 2679
eaaaatttga?ggacaaaccc?agattgggat?tggaatatgc?actgtaaatc?aaatttttct 2739
tatctacaaa?gactaatgta?aaaatgattt?tttcttctgt?gcctgattaa?attaactgtg 2799
gtttttaata?taaatattta?ttggtgtgct?ttgggagaaa?aattatcttt?tcttgaaaga 2859
agttatcaaa?gcaaatttat?tatcttcaca?agttaatggg?agaatgtggt?tttgattctg 2919
ggtgtttgaa?ttgtgtaaac?acacagcttc?cttgtgtgaa?gagagttgtc?ctgtgctcct 2979
tctactgtac?tttttttatt?ttttaatagg?aaagtgatgt?gcttcagaga?agggttcctc 3039
cttaatgttg?aggtttttta?aaaataaaaa?ttaatgttga?ggttttttaa?aaataaaaat 3099
actgttttta?agctctttcc?ttattggttt?gcatgattat?tggcatctgc?ttacgatggc 3159
tttaatcact?cagagctaat?ggcacctttc?ctatttagcc?tcattttaga?atgcagtcaa 3219
cacatgtaga?cttttcacaa?ataaatgaac?aacttcaaaa?aaaaaaaaaa?aaaa 3273
<210>2
<211>183
<212>PRT
<213>Homo?sapiens
<400>2
Met?Trp?Lys?Leu?Gly?Arg?Gly?Arg?Val?Leu?Leu?Asp?Glu?Pro?Pro?Glu
1 5 10 15
Glu?Glu?Asp?Gly?Leu?Arg?Gly?Gly?Pro?Pro?Pro?Ala?Ala?Ala?Ala?Gln
20 25 30
Ala?Gln?Val?Gln?Gly?Ala?Ser?Phe?Arg?Gly?Trp?Lys?Glu?Val?Thr?Ser
35 40 45
Leu?Phe?Asn?Lys?Asp?Asp?Glu?Gln?His?Leu?Leu?Glu?Arg?Cys?Lys?Ser
50 55 60
Pro?Lys?Ser?Lys?Gly?Thr?Asn?Leu?Arg?Leu?Lys?Glu?Glu?Leu?Lys?Ala
65 70 75 80
Glu?Lys?Lys?Ser?Gly?Phe?Trp?Asp?Asn?Leu?Val?Leu?Lys?Gln?Asn?Ile
85 90 95
Gln?Ser?Lys?Lys?Pro?Asp?Glu?Ile?Glu?Gly?Trp?Glu?Pro?Pro?Lys?Leu
100 105 110
Ala?Leu?Glu?AspIle?Ser?Ala?Asp?Pro?Glu?Asp?Thr?Val?Gly?Gly?His
115 120 125
Pro?Ser?Trp?Ser?Gly?Trp?Glu?Asp?Asp?Ala?Lys?Gly?Ser?Thr?Lys?Tyr
130 135 140
Thr?Ser?Leu?Ala?Ser?Ser?Ala?Asn?Ser?Ser?Arg?Trp?Ser?Leu?Arg?Ala
145 150 155 160
Ala?Gly?Arg?Leu?Val?Ser?Ile?Arg?Arg?Gln?Ser?Lys?Gly?His?Leu?Thr
165 170 175
Asp?Ser?Pro?Glu?Glu?Ala?Glu
180
Claims (9)
1. a nf TDRP1 is characterized in that, the aminoacid sequence of this nf is shown in SEQ ID NO2.
2. nf as claimed in claim 1 is characterized in that, the nucleotide sequence of this nf is shown in SEQ ID NO 1.
3. the preparation method of the described nf of claim 1 is characterized in that, according to the aminoacid sequence synthetic of SEQ ID NO 2.
4. the cloning vector or the expression vector that contain the nucleotide coding sequence of having the right to want 1 described nf.
5. the polyclonal antibody of the described nf of claim 1.
6. the described Polyclonal Antibody Preparation method of claim 5 is characterized in that this method comprises the steps:
(1) obtains the complete encoding sequence gene;
(2) with gene and prokaryotic expression carrier reorganization;
(3) recombinant expression vector is transduceed prokaryotic expression system;
(4) induce the expression of TDRP1 with IPTG, the TDRP1 after the expression is through gsh affinity chromatography purifying in addition, and excises the GST label with zymoplasm from fusion rotein and obtain the good TDRP1 albumen of purifying;
(5) immunity of the TDRP1 behind purifying New Zealand white rabbit obtains TDRP1 polyclonal antibody serum.
7. preparation method as claimed in claim 6 is characterized in that described prokaryotic expression carrier is pGEX-4T-2.
8. preparation method as claimed in claim 6 is characterized in that, described prokaryotic expression system is ROSSET (DE3) or BL21 (DE3).
9. the application of the described nf of claim 1 is characterized in that, this nf is used to regulate and control spermatogeny or testicualr development process.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010100230452A CN102127163A (en) | 2010-01-20 | 2010-01-20 | Cloning, protein expression, polyclonal antibody preparation and use of nuclear factor TDRP1 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010100230452A CN102127163A (en) | 2010-01-20 | 2010-01-20 | Cloning, protein expression, polyclonal antibody preparation and use of nuclear factor TDRP1 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102127163A true CN102127163A (en) | 2011-07-20 |
Family
ID=44265463
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010100230452A Pending CN102127163A (en) | 2010-01-20 | 2010-01-20 | Cloning, protein expression, polyclonal antibody preparation and use of nuclear factor TDRP1 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102127163A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107998397A (en) * | 2017-12-07 | 2018-05-08 | 复旦大学附属华山医院 | Application of the EMC10 albumen as biomarker in male sterility is diagnosed |
| WO2020201462A1 (en) * | 2019-04-03 | 2020-10-08 | Queen Mary University Of London | Treatment and diagnosis of mental disorders |
-
2010
- 2010-01-20 CN CN2010100230452A patent/CN102127163A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107998397A (en) * | 2017-12-07 | 2018-05-08 | 复旦大学附属华山医院 | Application of the EMC10 albumen as biomarker in male sterility is diagnosed |
| CN107998397B (en) * | 2017-12-07 | 2020-09-04 | 复旦大学附属华山医院 | Application of EMC10 protein as biomarker in diagnosis of male infertility |
| WO2020201462A1 (en) * | 2019-04-03 | 2020-10-08 | Queen Mary University Of London | Treatment and diagnosis of mental disorders |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2740417B2 (en) | Preparation method of human nerve growth factor by genetic recombination | |
| Shapiro et al. | Expression of Met-(− 1) angiogenin in Escherichia coli: conversion to the authentic< Glu-1 protein | |
| CN100354417C (en) | Novel serine protease genes related to DPPIV | |
| CN102121023B (en) | Mutant human plasminogen kringle5, preparation method and application thereof | |
| JPH06217778A (en) | Arrangement of human mk gene and protein | |
| US7666641B2 (en) | Canine COX-2 proteins and uses thereof | |
| CN102671185A (en) | Application of scolopendra mutilans neurotoxin peptide omega-SLPTX-Ssmla | |
| CN102127163A (en) | Cloning, protein expression, polyclonal antibody preparation and use of nuclear factor TDRP1 | |
| CN101497863B (en) | Method for preparing N-terminated acetylated thymosin alpha 1 and special engineering bacteria therefor | |
| JPH1017600A (en) | Blood platelet activating factor acetyl hydrolase and its gene | |
| CN1296967A (en) | Polypeptide-human deaf related gene 14 and polynucleotide for coding said polypeptide | |
| Chen et al. | Molecular cloning of a plasma membrane Ca2+ ATPase (PMCA) from Y-organs of the blue crab (Callinectes sapidus), and determination of spatial and temporal patterns of PMCA gene expression | |
| Witkowski et al. | Molecular cloning and sequencing of a cDNA encoding the acyl carrier protein and its flanking domains in the mammalian fatty acid synthetase | |
| CN1297916A (en) | Human bromo structure domain-containing protein 95 as one new kind of polypeptide and polynucleotides encoding this polypeptide | |
| WO2001038371A1 (en) | A NOVEL POLYPEPTIDE-HUMAN GLUTAMATE tRNA SYNTHETASE 58 AND THE POLYNUCLEOTIDE ENCODING SAID POLYPEPTIDE | |
| KR102631925B1 (en) | Development of novel fgf21 variants, production techniques and uses thereof | |
| JP4805945B2 (en) | Process for producing protein phosphatase type 2A enzyme heterodimer derivative | |
| JP3803978B2 (en) | Glucagon-degrading enzyme, gene encoding the same, and antibody against the glucagon-degrading enzyme | |
| WO2001038545A1 (en) | A novel polypeptide, a human acetyl galactosyl transferase 45 and the polynucleotide encoding the polypeptide | |
| CN101481414A (en) | Ovary function associated protein, and preparation and use thereof | |
| US20040005658A1 (en) | Novel polypeptide-human an1-like protein 16 and the polynucleotide encoding the same | |
| CN109609532A (en) | A kind of production method of anti-oxidant fusion protein | |
| CN1303931A (en) | Novel polypeptide-XRN-100 and polynucleotide coding said polypeptide | |
| WO2006054666A1 (en) | Chick lif protein and utilization thereof | |
| JP2000201684A (en) | Sodium channel scn8 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20110720 |