Kev Tshawb Fawb Txog Kev Tshaj Tawm Ntawm Cov Txheej Txheem Siab Siab Fermentation Lysate Ntawm Recombinant Escherichia Coli

 

Txhawm rau txo qhov cuam tshuam ntawm cov khoom siv dej rau chromatography thiab txhim kho kev rov qab los ntawm lub hom phiaj cov protein, cov txheej txheem nthuav dav ntawm kev ua kom siab ceev fermentation lysate ntawm ob hom recombinant Escherichia coli tau kawm. Hom BL21-HP1036 thiab BL21-palA qhia cov noob tseem ceeb ntawm Streptococcus suis thiab Haemophilus parasuis raug xaiv. Cov txheej txheem tangential ndlwg, cov txheej txheem centrifugation ceev thiab txheej txheem txheej txheem membrane cassette tau siv los ntsuas cov protein tawm los ntawm turbidity thiab electrophoresis nrhiav kom tau.

 

Cov txiaj ntsig tau pom tias BL21-HP1036, BL21-palA high density fermentation lysate qhia cov noob tseem ceeb ntawm Streptococcus suis thiab Haemophilus parasuis tau qhia meej los ntawm daim nyias nyias cov ntaub ntawv pom thiab centrifugation, thiab cov protein ntau tshaj 80%, uas tuaj yeem txo cov chromatographic cuam tshuam thiab txhim kho tus nqi rov qab.

 

Taw qhia

Tam sim no, Streptococcus suis (SS) thiab Haemophilus parasuis (HPS) yog cov kab mob uas muaj kab mob ntau tshaj plaws hauv npua teb, uas ua rau muaj kev poob zoo rau kev lag luam npua. SS tuaj yeem muab faib ua tag nrho ntawm 35 serotypes 1-34 thiab 1/2, thiab HPS tuaj yeem muab faib ua 1-15 serotypes, thiab ob feem ntau sib xyaw, feem ntau ua rau swine polyserositis, mob caj dab, mob meningitis, endocarditis. , kab mob septicemia thiab bronchitis.

 

Vim muaj coob tus serotypes ntawm Streptococcus suis thiab Haemophilus parasuis, kev tiv thaiv kev tiv thaiv ntawm cov serotypes sib txawv tsis muaj zog, thiab cov tshuaj tiv thaiv kab mob yuav nce ntxiv thaum kho nrog tshuaj tua kab mob, uas yuav cuam tshuam rau kev kho mob.

 

Cov tshuaj tiv thaiv tau dhau los ua ib txoj hauv kev zoo los tiv thaiv SS thiab HPS. Muaj cov tshuaj tiv thaiv tsis muaj zog, cov tshuaj tiv thaiv tsis zoo, cov tshuaj tiv thaiv nyob, cov tshuaj tiv thaiv subunit thiab cov tshuaj tiv thaiv kab mob, tab sis cov tshuaj tiv thaiv tsis ua haujlwm thiab cov tshuaj tiv thaiv attenuated feem ntau yog nyob rau hauv kev ua lag luam, thiab lawv cov kev tiv thaiv kev tiv thaiv yog qhov zoo, tab sis kev tiv thaiv hla tsis pom tseeb. Cov tshuaj tiv thaiv Subunit yog ib hom tshuaj tiv thaiv kab mob streptococcus suis tshiab, uas yog ib hom tshuaj tiv thaiv tshiab uas muaj kev tiv thaiv kab mob sib kis thiab tus nqi qis. Kev tshawb nrhiav thiab kev tsim cov tshuaj tiv thaiv subunit ntawm Streptococcus suis thiab Haemophilus parasuis muab kev daws teeb meem zoo tshaj plaws rau kev tiv thaiv thiab tswj cov teeb meem ntawm Streptococcus suis thiab Haemophilus parasuis uas plague kev lag luam npua hauv Suav teb.

 

Streptococcus suis thiab Haemophilus parasuis subunit tshuaj tiv thaiv yog raug rau siab ceev fermentation los ntawm recombinant Escherichia coli. Kev nthuav qhia ntawm lysate muaj txiaj ntsig zoo rau cov txiaj ntsig ntawm cov hom phiaj protein, tab sis muaj qee cov lus ceeb toom cuam tshuam. Yuav ua li cas kom paub meej cov kauj ruam ntawm fermentation thiab purification txheej txheem yog ib qho tseem ceeb ua hauj lwm. Nyob rau hauv daim ntawv no, recombinant E. coli BL21-HP1036, BL21-palA high-density fermentation raws li cov khoom, los kawm yuav ua li cas kom txo tau cov turbidity ntawm clarifying tov, txhim kho cov protein yield, rau Streptococcus suis subunit txhaj tshuaj tiv thaiv txheej txheem fermentation kom ua tiav kev lag luam crushing thiab clarification technology los muab theoretical hauv paus thiab kev txhawb nqa.

 

1. Cov ntaub ntawv thiab cov txheej txheem

1.1 Cov khoom siv

 

1.1.1 Cov

Streptococcus suis thiab Haemophilus parasuis hom BL21-HP1036, BL21-pa-lA

 

1.1.2 Main media thiab reagents

Poov xab extract thiab porpoise; Sodium chloride; Kanamycin, IPTG; formaldehyde los ntawm; SDS-PAGE gel cov khoom siv.

 

1.1.3 Cov khoom siv ntsuas tseem ceeb

High ceev fermenter; Kev kub ceev centrifuge; Electrophoresis apparatus; Ultraviolet gel duab system; Ultrasonic crusher; Me me kev kub ceev centrifuge; Ultraviolet spectrophotometer; Qhov kub thiab txias tsis tu ncua; Affinity chromatography khoom siv.

 

1.2 Cov txheej txheem

 

1.2.1 High-density fermentation kab lis kev cai

Cov kev tsim nyog recombinant coliform hom BL21-HP1036 thiab BL21-pa-lA tau inoculated ntawm nruab nrab hluavtaws ntawm 2% (V/V) thiab kab lis kev cai ntawm 37 degree rau 35-40 h.

 

1.2.2 Recombinant protein induced qhia

Thaum cov tshuaj fermentation OD {{0}}±{4}}.1, qhov induction pib, qhov induction kub yog 35 degree ± 0.5 degree, thiab kab lis kev cai xaus tom qab 10h ntawm induction.

 

1.2.3 High-pressure homogeneous crushing thiab centrifugation

Cov kab mob ua kua uas dhau qhov kev sim ua kom tsis muaj zog yog crushed los ntawm kev kub siab homogenizer rau cell cracking feem, nrog lub siab ntawm 100 ~ 150 MPa. Tom qab crushing, cov kua crushed tau khaws cia ntawm qhov kub thiab txias.

Lub supernatant tau txais los ntawm centrifuge nrog kev kub ceev, centrifugal ceev: 15000 rpm, kub tswj: 10 degree ~ 15 degree, txhaj ceev: 0.5 ~ 1 L / min, raws li, tus supernatant tau sau, thiab cov supernatant tau ntim nrog cov thawv uas tshem tawm endotoxin.

 

1.2.4 Microfiltration membrane cassette clarification

Cov daim nyias nyias tau ntxuav nrog 10L ntawm cov dej huv hauv ib qho kev coj, thiab kev lim dej tangential tau ua tiav. 300 mL ntawm cov kab mob tawg tom qab centrifugation tau muab los ntawm cov raj xa dej centrifuge thiab qhia meej nrog 0.45um microfiltration membrane cassette. Turbidity tau ntsuas los ntawm kev ua piv txwv, thiab turbidity tom qab centrifugation, turbidity ntawm clarified kua nyob rau hauv lub microfiltration membrane cassette thiab turbidity ntawm cov concentrated kua nyob rau hauv lub microfiltration membrane cassette tau muab piv. Cov ntsiab lus protein ntawm cov qauv tau kuaj pom los ntawm chromatography thiab electrophoresis los ntsuas cov protein tawm los.

 

Kauj Ruam 2: Cov txiaj ntsig

2.1 Cov ntaub ntawv qhia meej thiab cov txiaj ntsig ntawm BL21-HP1036 thiab BL21-palA protein

Cov txiaj ntsig hauv FIG. 1 qhia tau hais tias cov turbidity ntawm BL21-HP1036 thiab BL21-palA recombinant Escherichia coli crushing kua poob los ntawm 4 500 NTU thiab 4 000 NTU rau 1970 NTU thiab 520 NTU, raws li, Tom qab lub raj centrifugation. Lub turbidity ntawm BL21-HP1036 thiab BL21-palA recombinant Escherichia coli raug txo mus rau 25 NTU thiab 1.28 NTU los ntawm microfiltration membrane cassette txoj kev. Nws tuaj yeem pom tau tias kev siv cov microfiltration membrane cassette tangential flow filtration thiab optimized open flow channel muaj cov txiaj ntsig zoo ntawm kev txo cov endotoxin, viscosity thiab turbidity ntawm cov kua chromatographic dua li cov tsoos centrifuge pom, thiab tuaj yeem txo qhov tawg recombinant E. coli heteroprotein. thiab nucleic acid.

 

2.2 Protein rov qab ntawm BL21-HP1036 thiab BL21-palA qauv

Table 1 qhia tau hais tias recombinant Escherichia coli BL21-HP1036 protein muaj nyob rau hauv ob qho tib si microfiltration clarified tov thiab cov concentrated supernatant them los ntawm microfiltration membrane, thiab tag nrho cov protein tawm los tom qab rov qab cov protein los ntawm microfiltration clarified tov thiab cov concentrated supernatant. yog 88.5%. BL21-palA protein kuj muaj nyob rau hauv cov microfiltration exudate clarified tov thiab cov supernatant ntawm cov concentrated tov them los ntawm microfiltration membrane cassette. Tag nrho cov txiaj ntsig ntawm BL21-PALa protein yog 81.5%.

 

3. Cov lus xaus

Hauv daim ntawv no, cov kab mob uas qhia txog cov noob tseem ceeb BL{{0}}HP1036 thiab BL21-palA ntawm Streptococcus suis thiab Haemophilus parasuis feem cuam tshuam txog kev sib xyaw ua ke siab thiab dhau 0.45 Lub turbidity ntawm cov khoom ua kua yog txo qis los ntawm um microfiltration membrane cassette txheej txheem, qhia tau hais tias tangential flow filtration technology nrog microfiltration membrane cassette zoo dua li nrog high-ceev centrifuge, thiab yuav txo tau cov impurity protein thiab nucleic acid ntawm cov tawg recombinant Escherichia coli.

 

Tom qab sau cov kua ntshiab rau hauv ib qho microfiltration membrane cassette, muaj 62.9% Raws li 37.1% ntawm lub hom phiaj protein ntawm BL21-HP1036 yog microfiltration concentrate los yog muaj nyob rau hauv microfiltration membrane cassette thiab lwm yam poob. , ib txoj kev ob-kauj ruam tau txais kev pom tseeb. Hauv thawj kauj ruam, 0.45um microfiltration membrane cassette tau siv los qhia meej thiab sau cov exudate; Nyob rau hauv lub thib ob kauj ruam, cov seem concentrated kua tau sau tom qab tube centrifugation, thiab ob kauj ruam clarified kua tau ua ke raws li cov qauv chromatographic Cov turbidity tus nqi ntawm BL21-palA fermentation kua tsis nce ntau, cov protein yield tuaj yeem ncav cuag. ntau dua 85.5%, thiab cov protein tawm los ntawm BL21-Pala fermentation kua tuaj yeem ncav cuag 81.5% tom qab kev kho ob-kauj ruam.

 

Raws li kev xaiv cov microfiltration membrane cassette, Guidling Technology tuaj yeem muab cov kev daws teeb meem zoo rau koj. Peb cov microfiltration membrane cassette thiab ultrafiltration membrane cassette series cov khoom siv polyether sulfone ua cov cab kuj, tshwj xeeb tshaj yog tsim rau biotechnology. Peb cov polyether sulfone daim nyias nyias muaj qhov zoo ntawm kev ua haujlwm siab, tsis tshua muaj protein adsorption, siab txhua yam muaj zog, cuam tshuam tsis kam thiab lwm yam. Peb muab rau koj nrog tshwj xeeb membrane clamps los ntsuas qhov siab ntawm qhov nkag thiab qhov hluav taws xob hauv lub sijhawm, thiab tuaj yeem muab kev mus ntsib raws li koj xav tau los kho koj qhov tshwj xeeb ultrafiltration / microfiltration system. Xws li ib tug me me tangential flow system yuav siv tau rau me me, pilot thiab me-scale ntau lawm, thiab yuav ua tau tag nrho linear amplification kom tau raws li koj cov kev cai xeem thiab ntau lawm yuav tsum tau.

Hais txog Guidling

Guidling Technology yog lub teb chaws high-tech enterprise tsom rau biopharmaceuticals, cell culture, purification thiab concentration ntawm biomedicine, kuaj mob thiab muaj kua. Peb tau ua tiav cov khoom siv lim dej centrifugal, ultrafiltration & microfiltration cassettes, cov kab mob lim, TFF system, qhov tob lim, hollow fiber, thiab lwm yam. Uas tau raws li daim ntawv thov scenarios ntawm biopharmaceuticals, kab lis kev cai ntawm tes, thiab lwm yam. Peb cov membranes thiab cov lim dej lim tau dav siv hauv kev sib xyaw, kev rho tawm thiab kev sib cais ntawm kev pom ua ntej, microfiltration, ultrafiltration thiab nanofiltration. Peb cov kab ntau cov khoom, los ntawm me me, ib leeg-siv kuaj pom mus rau ntau lawm filtration systems, sterility kuaj, fermentation, cell kab lis kev cai thiab lwm yam, ua tau raws li cov kev xav tau ntawm kev sim thiab ntau lawm. Guidling Technology tab tom tos ntsoov rau kev koom tes nrog koj!