Kisspeptinyog ib qho me me molecule peptide tsim los ntawm 54 amino acid residues nrog ib qho hnyav molecular ntawm kwv yees li 6000 Daltons. Nws cov amino acid ib ntus tau txais txiaj ntsig zoo hauv cov tsiaj txhu, uas txhais tau hais tias nws muaj cov qauv zoo sib xws hauv ntau hom. Hauv tib neeg, cov amino acid sequence ntawm Kisspeptin yog H-Phe Gly Gly Leu Ser Arg Arg Al Glu Leu Ser Arg Arg Al Glu Leu Ser Arg Arg Al Glu Leu Ser Eu Ser Arg Al Glu Eu Ser Arg. Encoded los ntawm Kiss1 noob, cov noob no yog thawj zaug hloov mus rau hauv Kiss1 protein precursor, uas tau dhau los ua cov txheej txheem thiab kev sib txuas kom kawg tsim Kisseptin paub tab. Kev degradation ntawm Kisspeptin feem ntau yog ua los ntawm peptidases, uas zom nws mus rau hauv me me fragments los yog ib tug neeg amino acids. Nws plays lub luag haujlwm tseem ceeb hauv kev ua me nyuam. Nws suav hais tias yog ib qho tseem ceeb ntawm gonadotropin tso tawm cov tshuaj hormones (GnRH) tso tawm uas tuaj yeem txhawb kev tso tawm ntawm gonadotropins, yog li txhawb kev loj hlob thiab ovulation ntawm cov kab mob. Tsis tas li ntawd, Kisspeptin tseem koom nrog hauv kev tswj hwm lwm cov txheej txheem physiological, xws li kev xav, kev nco, thiab kev txawj ntse.
Kisspeptin peptide, tseem hu ua Kiss1 peptide lossis RFRP-1 peptide, yog ib qho neuropeptide pom hauv tib neeg lub cev. Hauv chav kuaj, cov txheej txheem hauv qab no feem ntau siv los ua ke Kisspeptin:
Tshuaj synthesis:
Tshuaj synthesis yog txoj kev siv ntau tshaj plaws rau kev sib txuas ntawm Kisspeptin hauv chav kuaj. Txoj kev no suav nrog ntau yam tshuaj tiv thaiv, xws li condensation, deprotection, thiab desalination. Ntawm lawv, cov kauj ruam tseem ceeb yog tsim cov peptide bonds ntawm cov amino acids, feem ntau yog siv classical coupling agents xws li EDC (1-ethyl-3- (3-dimethylaminopropyl) - carbodiimide) los yog BOP ( benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate). Qhov kom zoo dua ntawm cov tshuaj synthesis yog tias nws tuaj yeem tau txais qhov siab-purity Kisspeptin, tab sis qhov tsis zoo ntawm txoj kev no yog tias nws yuav tsum muaj cov kauj ruam ntawm kev sim thiab kev sim nruj heev, thaum cov txiaj ntsig qis.
Cov kauj ruam tshwj xeeb yog cov hauv qab no:
1. Npaj cov khoom pib. Qhov no suav nrog cov amino acids uas yuav tsum tau muaj, cov activators (xws li EDC lossis BOP), cov tshuaj tiv thaiv kev tiv thaiv (xws li trifluoroacetic acid lossis hydrobromic acid), nrog rau lwm cov tshuaj reagents tsim nyog thiab cov tshuaj tsis haum.
2. Nyob rau hauv cov dej tsis muaj dej thiab tsis muaj oxygen, yaj cov amino acids uas yuav tsum tau ua hauv cov kuab tshuaj uas tsim nyog xws li dimethylformamide (DMF) lossis N, N-dimethylacetamide (DMA).
3. Ntxiv cov activator uas yuav tsum tau ua (xws li EDC lossis BOP) thiab do hauv chav sov rau ib lub sijhawm los tsim peptide bonds.
4. Ntxiv deprotective reagents (xws li trifluoroacetic acid los yog hydrobromic acid) rau cov tsim peptide bonds kom tshem tawm cov amino tiv thaiv pawg.
5. Ntxiv cov pab pawg tiv thaiv uas yuav tsum tau muaj (xws li Boc lossis Fmoc) los tiv thaiv cov amino pawg tshiab.
6. Rov ua cov kauj ruam saum toj no kom txog thaum tag nrho cov amino acids uas yuav tsum tau sib txuas.
7. Ntxiv cov kev hloov pauv ntawm cov saw hlau uas xav tau thiab / lossis cov cim rau cov saw peptide.
8. Thaum kawg, cov tshuaj tiv thaiv deprotection tau ua kom tshem tawm tag nrho cov pab pawg tiv thaiv thiab tau txais Kisspeptin purified.
Cov saum toj no yog ib qho yooj yim tshuaj synthesis txoj kev, thiab cov kauj ruam tshwj xeeb yuav txawv nyob ntawm seb tus Kisspeptin ib ntus thiab yuav tsum tau hloov kho. Thaum lub sij hawm tag nrho cov txheej txheem synthesis, nws yog ib qho tsim nyog yuav tsum nruj me ntsis tswj cov kev sim, xws li hnyav, kub, pH, lub sij hawm, thiab siab, kom ntseeg tau tias qhov kev vam meej ntawm cov tshuaj tiv thaiv thiab siab purity ntawm cov khoom. Nyob rau tib lub sijhawm, nws yog ib qho tsim nyog yuav tsum tau ua tib zoo saib xyuas kev nyab xeeb ntawm cov tshuaj tiv thaiv thiab tsis txhob siv cov tshuaj txaus ntshai thiab kev ua haujlwm.
Genetic engineering synthesis:
Genetic engineering synthesis yog ib txoj hauv kev ua tau zoo, nrawm, thiab kev lag luam rau kev sib txuas ntawm Kisspeptin. Txoj kev no siv cov cuab yeej siv tshuaj genetic engineering los qhia txog cov protein ua ntej ntawm Kisspeptin hauv cov kab mob xws li Escherichia coli lossis poov xab, thiab tom qab ntawd ua tiav tom qab ua tiav kom tau txais Kisspeptin paub tab.
Cov hauv qab no yog cov txheej txheem yooj yim:
1. Gene cloning: Ua ntej, nws yog ib qho tsim nyog kom tau txais cov noob caj noob ces ntawm Kisspeptin. Qhov no tuaj yeem tau txais los ntawm cov ntaub so ntswg lom neeg los ntawm RT PCR, genome sequencing, lossis lwm yam txheej txheem gene cloning.
2. Vector xaiv: Tom ntej no, koj yuav tsum xaiv ib lub vector los tso cov noob kab ke ntawm Kisspeptin. Qhov no feem ntau yog cov kab mob uas tsis muaj kab mob plasmid lossis kab mob kab mob. Lub vector yog tsim los ntxig cov noob Kisspeptin thiab coj mus rau hauv lub cell.
3. Gene transformation: Ntxig cov noob Kisspeptin rau hauv vector, thiab tom qab ntawd hloov cov kab no (gene + vector) rau hauv cov kab mob engineering xws li Escherichia coli lossis poov xab.
4. Kev nthuav qhia: Hauv cov kab mob engineering, Kisspeptin gene yog "nyeem" thiab coj cov protein synthesis. Cov proteins no feem ntau yog txuas nrog cov tshuaj tshwj xeeb rau cov txheej txheem purification tom ntej.
5. Tom qab ua tiav: Cov Kisspeptin precursor proteins uas tsim los ntawm cov kab mob engineered tuaj yeem sau thiab ua kom huv los ntawm cov kauj ruam xws li cell fragmentation, centrifugation, thiab dialysis.
Qhov kom zoo dua ntawm txoj kev no yog tias nws tuaj yeem tsim tau ntau npaum li cas ntawm Kisspeptin nyob rau lub sijhawm luv luv, thiab tus nqi kuj tsawg. Tsis tas li ntawd, vim yog siv cov kab mob me me, txoj kev tsim khoom no muaj kev cuam tshuam tsawg heev rau ib puag ncig. Txawm li cas los xij, qhov tsis zoo ntawm txoj kev no yog tias nws yuav tsum tau siv cov kab mob microorganisms thiab yog li yuav tsum muaj qee yam khoom siv hauv chav kuaj thiab kev txawj ntse.
Bioenzymatic hydrolysis:
Bioenzymatic hydrolysis yog ib txoj hauv kev los ua ke Kisspeptin siv enzyme catalysis. Txoj kev no siv cov enzymes tshwj xeeb los hloov Kisspeptin precursor proteins rau Kisspeptin paub tab.
Cov kauj ruam yooj yim rau kev sib txuas ntawm Kisspeptin los ntawm kev lom enzymatic hydrolysis:
1. Gene cloning thiab vector xaiv: Ua ntej, nws tseem yuav tsum tau txais cov noob kab ke ntawm Kisspeptin, thiab tom qab ntawd xaiv ib lub vector los ntxig nws.
2. Kev nthuav qhia: Ntxig cov noob Kisspeptin rau hauv vector, thiab tom qab ntawd hloov qhov complex (gene + vector) mus rau hauv cov kab mob engineering.
3. Protein synthesis: Hauv cov kab mob engineering, Kisspeptin gene yog "nyeem" thiab coj cov protein synthesis. Cov protein no feem ntau yog txuas nrog cov ntawv tshwj xeeb.
4. Bioenzymatic hydrolysis: Kev siv cov proteases tshwj xeeb, xws li subtilisin lossis trypsin, los hloov cov protein ua ntej mus rau Kisspeptin paub tab. Cov enzymes lom neeg muaj qhov tshwj xeeb thiab catalytic efficiency, yog li cov kauj ruam ntawm cov tshuaj tiv thaiv no tuaj yeem ua tiav sai thiab zoo.
5. Tom qab ua tiav: Los ntawm ntau cov kauj ruam xws li cell fragmentation, centrifugation, dialysis, thiab lwm yam., Kisspeptin yog thaum kawg sau thiab purified.
Qhov zoo ntawm txoj kev no yog tias nws tuaj yeem ua tiav cov synthesis ntawm Kisspeptin hauv lub sijhawm luv luv. Tsis tsuas yog lub synthesis ceev ceev, tab sis kuj lub catalytic efficiency ntawm lom enzymes yog tsis tshua muaj siab, uas yuav zoo heev txhim kho cov yield ntawm lub hom phiaj proteins. Lub caij no, cov enzymes lom neeg siv hauv enzymatic hydrolysis feem ntau muaj qhov tshwj xeeb thiab tuaj yeem ua tau zoo thiab ua haujlwm tau zoo hauv cov kab mob lom neeg. Yog li ntawd, txoj kev no yog ib puag ncig tus phooj ywg thiab muaj kev cuam tshuam me ntsis rau cov qauv thiab kev ua haujlwm ntawm lub hom phiaj protein. Txawm li cas los xij, txoj kev no kuj muaj qee qhov kev txwv, xws li kev nyuaj rau kev tau txais thiab npaj cov enzymes lom neeg, cov nqi siab, thiab kev xav tau rau kev tswj xyuas meej ntawm cov tshuaj tiv thaiv.
Cell kab lis kev cai:
Cell kab lis kev cai yog ib txoj kev ntawm kev sib txuas ntawm Kisspeptin hauv chav kuaj. Txoj kev no suav nrog kev tsim cov kab ntawm tes uas muaj cov noob caj noob ces Kisspeptin, thiab tom qab ntawd sau cov Kisspeptin secreted los ntawm kab lis kev cai nruab nrab. Tshwj xeeb, cov kab ke ntawm Kisspeptin yog thawj zaug tso rau hauv kab ntawm tes, ua raws li kab lis kev cai ntawm tes thiab kev ua kom zoo. Thaum kawg, Kisspeptin tau sau los ntawm kab lis kev cai nruab nrab. Qhov kom zoo dua ntawm kab lis kev cai ntawm tes yog tias nws tuaj yeem tsim tau ntau ntawm Kisspeptin, thiab kev ua haujlwm ntawm txoj kev no yog qhov yooj yim.
Hauv cov ntsiab lus, muaj ntau txoj hauv kev rau kev sib txuas ntawm Kisspeptin hauv chav kuaj, thiab txhua txoj kev muaj nws qhov zoo thiab qhov tsis zoo. Cov txheej txheem tsim nyog tuaj yeem xaiv rau kev sib xyaw ua ke raws li qhov xav tau tiag tiag. Ntawm lawv, chemical synthesis thiab genetic engineering synthesis yog cov kev siv ntau tshaj plaws, thaum enzymatic hydrolysis thiab cell kab lis kev cai yog lwm txoj kev xaiv.