IGF-1 LR3 Lyophilized,The freeze-dried long-acting insulin-like growth factor-1 LR3 formulation uses low-temperature vacuum drying technology to remove moisture, preventing peptide degradation or aggregation in liquid formulations and extending the shelf life (usually up to 2 years at -20 ℃). It can be directly used after reconstitution, reducing transportation and storage costs. At the same time, the freeze-drying process can remove impurities and improve the purity of the formulation (usually>95%). Txawm li cas los xij, kev siv sijhawm ntev - tuaj yeem ua rau receptor desensitization thiab xav tau kev tswj xyuas tsis tu ncua. Kev noj tshuaj ntau dhau tuaj yeem ua rau hypoglycemia, thiab cov ntshav qabzib yuav tsum tau saib xyuas.
Peb tsis tsuas yog muab cov hmoov ntshiab thiab cov tshuaj ntsiav, tab sis kuj muaj lwm yam tshuaj. Yog tias tsim nyog, thov hu rau peb pab neeg muag khoom.
Peb cov khoom los ntawm
IGF-1 LR3 COA
Gene cloning thiab qhia vector tsim
Lub gene tsim ntawmIGF-1 LR3 Lyophilizedyog raws li qhov sib lawv liag ntawm ntuj IGF-1, tab sis nrog cov kev hloov pauv tseem ceeb hauv qab no:
N-terminal extension: Ntxiv 13 amino acids (MFPAMPLSSLFFN) mus rau N-terminus ntawm IGF-1 kom ncua nws ib nrab-lub neej thiab txhim kho nws txoj kev lom neeg.
Amino acid hloov pauv: Hloov glutamic acid (Glu) ntawm txoj hauj lwm 3 ntawm IGF-1 nrog arginine (Arg), piv txwv li R3 hloov kho, kom txo tau kev sib khi muaj peev xwm nrog IGFBP thiab nce dawb concentration.
noob synthesis
Tau txais lub hom phiaj gene fragment los ntawm chemical synthesis lossis PCR amplification raws li tus tsim IGF-1 LR3 gene sequence. Cov tshuaj synthesis feem ntau siv cov khoom siv hluav taws xob-theem synthesis, maj mam ntxiv nucleotides, thiab thaum kawg synthesizing tiav cov noob tawg. PCR amplification siv paub IGF-1 sequences los tsim primers thiab amplify lub hom phiaj noob los ntawm PCR cov tshuaj tiv thaiv.
Xaiv qhov tsim nyog qhia vector yog ib kauj ruam tseem ceeb hauv kev qhia cov protein. Cov vectors uas nquag siv muaj xws li pET series (xws li pET30a), pGEX series, thiab lwm yam. Cov vectors no muaj kev txhawb nqa zoo, ntau qhov chaw cloning, thiab cov cim npe (xws li Nws cov cim npe), uas pab txhawb cov protein purification thiab nrhiav kom tau tom qab.
Piv txwv li, kev tsim kho cov kauj ruam ntawm kev qhia vector pET30a yog raws li hauv qab no:
Enzymatic cleavage: Siv cov kev txwv endonucleases xws li BamHI thiab XhoI los ua ob lub enzyme cleavage ntawm pET32a vector thiab IGF-1 LR3 gene fragment, ua tiav cov nplaum nplaum.
Kev sib txuas: Lub enzyme digested vector thiab gene fragment yog txuas los ntawm T4 DNA ligase los ua cov recombinant qhia vector pET32a-LR3-IGF1.
Kev hloov pauv: Hloov cov kab lus rov ua dua tshiab rau hauv E. coli hlwb (xws li DH5 ) thiab tau txais cov clones zoo los ntawm kev tshuaj xyuas tshuaj tua kab mob (xws li ampicillin).
Kev txheeb xyuas: Txheeb xyuas qhov tseeb ntawm qhov rov ua kom pom vector los ntawm txoj hauv kev xws li colony PCR, enzyme digestion identification, lossis sequencing.
Protein kev qhia
Kev xaiv hom kev qhia
Xaiv qhov kev qhia tsim nyog yog qhov tseem ceeb rau kev qhia cov protein. Cov kab mob feem ntau siv xws li Rosetta (DE3), BL21 (DE3), thiab lwm yam. Cov kab no muaj cov kab mob T7 RNA polymerase zoo thiab tuaj yeem nthuav qhia cov noob uas tau tsav los ntawm T7 txhawb nqa.
Noj Rosetta (DE3) ua piv txwv, nws cov yam ntxwv muaj xws li: muaj T7 RNA polymerase gene, induced los ntawm IPTG qhia. Nqa cov noob tsis tshua muaj codon tRNA tuaj yeem nthuav qhia cov noob exogenous uas muaj cov codons tsawg. Haum rau kev nthuav qhia cov proteins uas muaj tshuaj lom lossis nyuaj rau kev nthuav qhia cov proteins.
Optimization ntawm kev nthuav qhia tej yam kev mob
Cov xwm txheej ntawm kev qhia cov protein, xws li inducer concentration, induction kub, induction lub sij hawm, thiab lwm yam, muaj kev cuam tshuam loj rau cov protein ntau thiab zoo. Los ntawm kev ua kom zoo rau cov xwm txheej, kev qhia theem ntawm IGF-1 LR3 tuaj yeem nce ntxiv.
Inducing agent concentration: IPTG yog ib tug nquag siv inducing agent, nrog ib tug concentration feem ntau ntawm 0.1-1 mM. Txiav txim siab qhov pom zoo IPTG concentration los ntawm kev sim gradient kom ua tiav qib siab tshaj plaws ntawm cov protein.
Induction kub: Induction kub feem ntau yog xaiv ntawm 16-37 degree. Qhov ntsuas kub qis dua (xws li 16-20 degree) tuaj yeem pab txo qis cov protein degradation thiab suav nrog lub cev tsim, thiab ua kom cov qoob loo ntawm cov protein ntau.
Lub sij hawm induction: Lub sij hawm induction feem ntau yog nyob nruab nrab ntawm 2-24 teev, thiab lub sij hawm zoo induction yog txiav txim los ntawm kev soj ntsuam cov protein qhia theem los ntawm lub sij hawm sampling.
Protein expression detection
Kev nthuav qhia ntawmIGF-1 LR3 Lyophilizedtau kuaj pom los ntawm SDS-PAGE thiab Western blot txoj kev.
SDS-PAGE: Sau cov kab mob ua ntej thiab tom qab induction, ua ultrasonic fragmentation lossis tshuaj lysis, centrifuge cais cov supernatant thiab precipitate, thiab ua SDS-PAGE tsom xam cais. Los ntawm kev sib piv cov kev hloov pauv ntawm cov kab mob protein nyob rau hauv lub supernatant thiab precipitate ua ntej thiab tom qab induction, qhov kev qhia ntawm IGF-1 LR3 tau lees paub.
Western blot: Siv cov tshuaj tiv thaiv tshwj xeeb (xws li tiv thaiv Nws cov tshuaj tiv thaiv kab mob lossis tshuaj tiv thaiv IGF-1 cov tshuaj tiv thaiv) los ua cov tshuaj tiv thaiv kab mob ntawm SDS-PAGE cais cov protein kom paub meej ntxiv txog qhov qhia thiab qhov hnyav ntawm IGF-1 LR3.
Protein Purification
Cov kab mob lysis
Sau cov kab mob induced thiab lyse lawv kom tso cov proteins hauv cov cellular. Cov txheej txheem cracking feem ntau suav nrog ultrasonic fragmentation, siab -siab homogenization, tshuaj cracking, thiab lwm yam. Siv ultrasonic fragmentation ua piv txwv, cov kauj ruam yog raws li nram no:
Bacterial collection: Centrifuge induced expression bacterial solution to collect the bacterial cells, and resuspend in lysis buffer (xws li PBS or Tris HCl buffer).
Ultrasonic fragmentation: Cov kab mob raug tshem tawm yog muab tso rau hauv ib da dej dej khov thiab crushed siv lub ultrasonic disruptor, feem ntau yog siv ib tug luv luv -term ntau fragmentation txoj kev (xws li 10 vib nas this txhua lub sij hawm, 10 vib nas this interval, rov 10 zaug) kom tsis txhob muaj protein ntau denaturation vim overheating.
Kev sib cais centrifugal: Centrifuge lub crushed kab mob ncua kev kawm ntawv cais cov supernatant thiab precipitate. Lub supernatant muaj cov proteins soluble, thaum lub precipitate tej zaum yuav muaj xws li lub cev los yog cell debris.
Affinity chromatography
Vim lub fact tias recombinant IGF-1 LR3 feem ntau nqa ib tug Nws tag, nws yuav ua kom huv si siv nickel ion affinity chromatography (Ni NTA).
Kem npaj: Load Ni NTA packing rau hauv chromatography kem thiab sib npaug nrog equilibration buffer (xws li PBS muaj 20 mM imidazole).
Sample loading: Load lub supernatant ntawm cov kab mob lysate mus rau kab chromatography, tso cai rau IGF-1 LR3 nrog Nws tag los khi nrog npib tsib xee ions.
Ntxuav: Ntxuav cov chromatography kem nrog kev ntxuav tsis huv (xws li PBS muaj 50 mM imidazole) kom tshem tawm cov protein tsis muaj zog lossis tsis muaj zog.
Elution: Siv elution buffer (xws li PBS muaj 250 mM imidazole) los elute IGF-1 LR3 khi rau kab chromatography, thiab sau cov eluent.
Desalination thiab Concentration
IGF-1 LR3 tov tom qab elution muaj ib tug siab concentration ntawm imidazole, uas yuav tsum tau muab tshem tawm los ntawm desalination thiab concentration cov kauj ruam kom muaj protein ntau concentration.
Desalination: Siv cov kab desalination los yog lub hnab lim dej kom tshem tawm imidazole los ntawm eluent thiab hloov nrog cov tshuaj tsis haum (xws li PBS).
Concentration: Concentrate cov desalinated protein tov siv ultrafiltration hlab los yog centrifugal concentrators kom cov protein ntau concentration mus rau qhov xav tau.
Purity thiab kev kuaj ua haujlwm
Kev huv huv thiab kev ua haujlwm ntawm IGF purified-1 LR3 tau kuaj pom los ntawm SDS-PAGE, HPLC, lossis ELISA txoj kev.
SDS-PAGE: Txheeb xyuas qhov purity ntawm cov protein purified thiab paub meej tias tsis muaj cov impurity pom tseeb.
HPLC: Siv cov theem rov qab HPLC lossis qhov loj me me tsis suav nrog HPLC txhawm rau txheeb xyuas qhov purity thiab molecular hnyav faib ntawm cov protein.
ELISA: Enzyme linked immunosorbent assay (ELISA) yog siv los txheeb xyuas cov haujlwm lom neeg ntawm IGF-1 LR3, thiab nws qib kev ua haujlwm tau cuam tshuam los ntawm qib kev khi rau cov tshuaj tiv thaiv tshwj xeeb.
Kho kom qhuav txheej txheem
Freeze drying txoj cai
Lyophilization yog txheej txheem ziab uas tshem tawm cov dej noo los ntawm qhov qis -kub khov thiab nqus sublimation. Lub khov- txheej txheem ziab muaj peb theem: ua ntej khov, sublimation drying, thiab desorption ziab, uas tuaj yeem ua kom muaj kev ua haujlwm thiab ruaj khov ntawm cov protein.
Cia kom qhuav cov kauj ruam
Faib cov IGF purified-1 LR3 tov rau hauv lub fwj khov-qhuav los yog penicillin fwj, thiab muab tso rau hauv lub tshuab ziab khaub ncaws kom khov hauv qab -40℃kom khov cov tshuaj tag.
Maj mam nce qhov kub kom txog -20℃nyob rau hauv lub tshuab nqus tsev, cia cov dej khov rau sublime ncaj qha rau hauv dej vapor thiab tshem tawm feem ntau ntawm cov dej noo. Nyob rau theem no, nws yog ib qho tsim nyog los tswj tus nqi cua sov thiab lub tshuab nqus tsev kom tsis txhob muaj protein ntau los yog tawg.
Ntxiv qhov ntsuas kub mus rau 20-30℃kom tshem tawm cov dej seem seem thiab txo cov dej noo ntawm cov khoom khov-qhuav kom qis dua 1%. Nyob rau theem no, nws yog ib qho tsim nyog yuav tsum muaj lub tshuab nqus tsev siab kom ntseeg tau tias kev tshem tawm tag nrho ntawm cov dej noo.
Tom qab ziab kom tiav, ceev nrooj kaw lub freeze- lub raj mis kom qhuav los yog penicillin lub raj mis kom tsis txhob ya raws los ntawm kev nkag mus.
Freeze drying tiv thaiv
Txhawm rau tiv thaiv kev ua haujlwm thiab kev ruaj ntseg ntawm IGF-1 LR3 thaum lub sijhawm khov- ziab, feem ntau yuav tsum tau ntxiv khov- tiv thaiv kom qhuav. Common freeze-drying protectants muaj xws li:
- Carbohydrates: xws li alginate, sucrose, qabzib, thiab lwm yam, tuaj yeem tsim cov qauv iav thiab ua kom ruaj khov ntawm cov protein.
- Polymers xws li polyethylene glycol (PEG) thiab polyvinylpyrrolidone (PVP) tuaj yeem ua kom cov viscosity ntawm kev daws teeb meem thiab txo cov protein sib sau ua ke.
- Amino acids, xws li glycine thiab alanine, tuaj yeem ua raws li hydrogen daim ntawv cog lus pub dawb lossis txais, ua kom cov qauv ntawm cov protein.
- Surfactants xws li Tween 20, Tween 80, thiab lwm yam tuaj yeem txo cov adsorption ntawm cov proteins mus rau lub thawv phab ntsa thiab txhim kho cov nqi rov qab.
Noj trehalose ua piv txwv, nws cov nyiaj ntxiv feem ntau yog 1-10 npaug ntawm cov protein ntau, uas tuaj yeem tsim cov txheej txheem tiv thaiv thaum khov-qhuav kom tsis txhob muaj protein ntau.
Optimization ntawm Freeze Drying Process
Kev ua kom zoo ntawm khov- txheej txheem ziab yog qhov tseem ceeb rau kev txhim kho qhov zoo ntawmIGF-1 LR3 Lyophilized. Los ntawm kev kho qhov tsis zoo xws li qhov kub thiab txias ua ntej, sublimation ziab kub, ntsuas qhov ziab kom qhuav, thiab lub tshuab nqus tsev, qhov zoo tshaj plaws khov - ziab kom qhuav tuaj yeem ua tiav.
Pre freezing kub
Qhov ntsuas kub ua ntej khov yuav tsum qis dua qhov taw tes eutectic ntawm cov protein kom ntseeg tau tias ua kom tiav khov ntawm cov tshuaj. Feem ntau, kub hauv qab -40℃yog xaiv.
Sublimation drying kub
Lub sublimation drying kub yuav tsum qis dua qhov kub ntawm lub cev qhuav dej kom tsis txhob muaj protein ntau denaturation lossis ntim ntim. Feem ntau nyob ib ncig ntawm -20℃yog xaiv.
Kev ntsuas kub kom qhuav
Kev soj ntsuam kom qhuav qhov kub yuav tsum siab tshaj qhov kub sublimation ziab kom tshem tawm cov seem seem. Feem ntau, 20-30℃yog xaiv.
Nqus degree
Lub tshuab nqus tsev kawm ntawv yuav tsum tau khaws cia nyob rau theem qis (xws li<100 mTorr) to promote the sublimation and removal of moisture.
Cim npe nrov: igf-1 lr3 lyophilized, Tuam Tshoj igf-1 lr3 lyophilized manufacturers, lwm tus neeg
