Kev Tsim Kho Ntawm Recombinant Lactobacillus Casei Vaccine Ntawm PEDV Thiab Nws Cov Tshuaj Tiv Thaiv Kab Mob hauv nas
Sep 06, 2023
Abstract
Keeb kwm: Porcine epidemic diarrhea (PED) yog kab mob plab hnyuv loj uas tshwm sim los ntawm tus kab mob porcine epidemic diarrhea (PEDV) uas ua rau ntuav, raws plab, anorexia, thiab lub cev qhuav dej, uas tau ua rau muaj kev puas tsuaj loj thoob ntiaj teb. Tam sim no, tshuaj tiv thaiv kab mob tseem yog txoj hauv kev zoo tshaj plaws los tswj kev sib kis ntawm PEDs. Nyob rau hauv txoj kev tshawb no, peb tau tsim ib tug tshiab recombinant L. casei-OMP16-PEDVS strain qhia PEDVS protein ntawm PEDV thiab OMP16 protein ntawm Brucella abortus hom. Txhawm rau paub txog kev tiv thaiv kab mob ntawm cov tshuaj tiv thaiv kab mob L. casei-OMP16-PEDVS tus neeg sib tw tshuaj tiv thaiv, nws tau muab piv nrog BL21-OMP16-PEDVS-F, BL21-OMP{{ 9}} PEDVS, thiab BL21-PEDVS recombinant protein.
Cov txiaj ntsig: Cov txiaj ntsig tau pom tias peb tuaj yeem kuaj pom cov qib siab dua ntawm IgG, neutralizing antibody, IL-4, IL-10, thiab INF- hauv cov ntshav thiab IgA hauv cov quav ntawm L. casei-OMP16- PEDVS txhaj tshuaj tiv thaiv nas, uas tau qhia tias L. casei-OMP16-PEDVS cov tshuaj tiv thaiv tuaj yeem ua rau muaj kev tiv thaiv kab mob siab dua, tiv thaiv kab mob ntawm tes, thiab kev tiv thaiv mucosal.
Xaus: Yog li ntawd, L. casei-OMP16-PEDVS yog ib qho tshuaj tiv thaiv zoo rau kev tiv thaiv kab mob PEDV.
Keywords: PEDV, Lactobacillus casei vaccine, PEDV S protein, OMP16, Immune responses
cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob
Taw qhia
Porcine epidemic diarrhea (PED) yog tshwm sim los ntawm tus kab mob porcine epidemic diarrhea (PEDV) nrog cov tsos mob xws li raws plab, ntuav, anorexia, lub cev qhuav dej, thiab poob phaus hauv piglets [1, 2]. Npua ntawm txhua lub hnub nyoog tuaj yeem kis tau nrog cov tsos mob sib txawv thiab qhov kev tuag ntawm cov npua yog li 100% [3], uas tau ua rau muaj kev puas tsuaj loj thoob ntiaj teb. Txhawm rau tswj kev sib kis ntawm PEDV, ntau hom tshuaj tiv thaiv yog tsim, xws li txhuas hydroxide-adjuvanted inactivated vaccine, bivalent inactive voted Transmissible Gastroenteritis Virus (TGEV), thiab PEDV tshuaj tiv thaiv, thiab attenuated PEDV tshuaj tiv thaiv [4]. Txawm hais tias cov tshuaj tiv thaiv no ua lub luag haujlwm tseem ceeb hauv kev tswj hwm PEDs, lawv txhua tus muaj lawv qhov tsis xws luag. Cov tshuaj tiv thaiv kab mob tsis tuaj yeem ua kom tsis muaj zog tiv thaiv kab mob hauv lub cev, tshuaj tiv thaiv kab mob tsis zoo, thiab lawv tsis tuaj yeem tsim cov kab mob tshwj xeeb IgA cov tshuaj tiv thaiv kab mob ntawm cov kab mob mucosal. Yog li ntawd, nws yog ib qho tsim nyog thiab ceev nrooj los tsim cov tshuaj tiv thaiv tshiab los tswj PEDs.
Lactobacillus casei feem ntau suav hais tias yog ib hom kev nyab xeeb vector system rau kev xa cov antigens hauv qhov ncauj, nrog rau cov txiaj ntsig zoo rau kev noj qab haus huv ntawm tib neeg thiab tsiaj txhu [5]. Lub caij no, nws tuaj yeem siv los ua kev xa khoom los tswj cov T-helper cell teb thiab txhawb kev tso tawm ntawm IgAs tshwj xeeb rau kev tiv thaiv mucosal [6]. Ntawm qhov tod tes, cov tshuaj tiv thaiv Lactobacillus casei recombinant yooj yim dua rau kev tswj hwm, muaj feem tsawg dua ntawm cov tshuaj tiv thaiv kab mob siab, thiab raug nqi ntau dua piv nrog cov tshuaj tiv thaiv ib txwm muaj. Raws li cov lus ceeb toom, Lactobacillus casei recombinant txhaj tshuaj tau siv tau zoo hauv kev tiv thaiv thiab kev tswj hwm ntawm tib neeg papillomavirus, Streptococcus pneumonia, thiab Escherichia coli [7–9]. Kuj tseem muaj qee qhov kev sim zoo sib xws ntawm kev tsim cov tshuaj tiv thaiv PED. Cov kws tshawb fawb pom tau hais tias ib qho kev sib txuas ntawm Lactococcus lactis strain uas qhia txog qhov sib txawv ntawm porcine kab mob raws plab kab mob S1 noob tuaj yeem ua rau muaj qib siab ntawm IL-4 thiab IFN- hauv kev txhaj tshuaj nas [10]. Lactobacillus casei-based anti-PEDV tshuaj tiv thaiv uas qhia txog microfold cell-targeting peptide Co1 fused nrog COE antigen ntawm PEDV tuaj yeem ua rau muaj kev tiv thaiv kab mob zoo [11]. Txhawm rau txhim kho qhov ua tau zoo ntawm cov tshuaj tiv thaiv PEDV. Hauv txoj kev tshawb no, peb tsim cov tshuaj tiv thaiv Lactobacillus casei recombinant tshiab ntawm PED, uas tuaj yeem ua kom muaj zog mucosal, humoural, thiab cellular tiv thaiv kab mob PEDV los ntawm kev tswj qhov ncauj. PEDV, ib tug tswv cuab ntawm tsev neeg coronaviridae, muaj plaub cov qauv proteins uas muaj 150-220 kDa glycosylated spike (S) protein, 20-30 kDa membrane (M) protein, 7 kDa hnab ntawv (E) protein, 58 kDa nucleocapsid (N) protein [12]. Nyob ntawd, S protein tuaj yeem muab faib ua S1 (1–735 amino acid) thiab S2 (736-kawg amino acid) domains [13], thiab S1 protein suav nrog thaj tsam receptor-binding thiab lub ntsiab neutralizing epitopes [ 14] ib. Cov tshuaj tiv thaiv adjuvant ua raws li immunomodulator thiab tuaj yeem tsim thiab txhim kho lub cev tiv thaiv kab mob tiv thaiv kev sib koom ua ke antigens. OMP16 protein ntawm Brucella abortus strain tau txheeb xyuas los ua kom cov hlwb dendritic hauv vivo, ua rau lub cev tiv thaiv kab mob th1, thiab yog cov tshuaj tiv thaiv tus kheej-adjuvant [15–17]. Yog li, OMP16 protein tau muab tso rau hauv Lacto bacillus case recombinant vaccine hauv peb txoj kev tshawb fawb. Txog tam sim no, ob peb txoj kev tshawb fawb txog Lactobacillus casei recombinant tshuaj tiv thaiv ntawm PEDV tau tshaj tawm. Yog li, txoj kev tshawb fawb no yog tsim los tsim cov tshuaj tiv thaiv kab mob Lactobacillus casei tshiab, uas tuaj yeem muab cov tshuaj tiv thaiv kab mob zoo dua qub, tiv thaiv kab mob ntawm tes, thiab kev tiv thaiv mucosal los tiv thaiv kev kis ntawm PED.
Suav tshuaj ntsuab cistanche cog-Antitumor
Cov ntaub ntawv thiab cov txheej txheem
Cov kab mob kab mob, kab mob, kab lis kev cai, plasmids, thiab primers
Cistanche hmoov
Cov kab mob kab mob, plasmids, thiab primers siv nyob rau hauv txoj kev tshawb no yog teev nyob rau hauv Table 1. Tus qauv siv hom ntawm Lactobacillus casei ATCC 393 yog kab lis kev cai nyob rau hauv de Man Rogosa thiab Sharpe (MRS) broth ntawm 37 degree [18]. Lub BL21 (DE3) thiab DH5 tau coj mus rau hauv Luria-Bertani (LB) nruab nrab ntawm 37 degree [19]. Cov recombinant Lactobacil lus casei, BL21 (DE3), thiab DH5 hom kab mob tau coj mus rau hauv cov nruab nrab sib xws nrog cov tshuaj tua kab mob kom raug, raws li. Brucella abortus tau loj hlob hauv Tryptic Soy Broth (TSB) lossis Tryptic Soy Agar (TSA) nruab nrab (Difco Laboratories, Detroit, MI, USA) ntawm 37 degree. Cov kab mob Vero kis tau tus kab mob PEDV tau coj mus kuaj hauv DMEM (Gibco, Langley, VA, USA) ntxiv nrog 10 ug / mL trypsin (Gibco, Langley, VA, USA) [20]. Lub pVE5523 thiab pET28a (+) plasmids tau qhia vectors ntawm Lactobacillus casei thiab Escherichia coli, raws li.
Table 1 Cov yam ntxwv ntawm cov kab mob kab mob, plasmids, thiab primers siv hauv txoj kev tshawb no
Kev tsim kho ntawm recombinant Lactobacillus casei thiab BL21 hom
Lub recombinant qhia plasmids tau tsim raws li cov plasmids thiab primers nyob rau hauv Table 1. Thaum xub thawj, ib feem ntawm PEDV S gene (493–708 amino acid), ib feem ntawm PEDV S' noob (493–708 amino acid), thiab ib feem ntawm Brucella abortus OMP16 noob (26–168 amino acid) tau nthuav dav siv cov primer khub PEDVS-F1/R1, PEDVS-F2/R2, thiab OMP16-F/R, raws li. Tom qab ntawd, txoj kev sib tshooj txuas ntxiv tau siv nyob rau hauv OMP16 thiab PEDVS 'fragments los tsim ib feem tshiab OMP16-PEDVS nrog linker polypeptide (GGGGSGGGGGGGGGGS) daig hauv nruab nrab. Tom qab ntawd, cov fragments PEDVS tau muab tso rau hauv pET28a plasmid nrog EcoRI/XbaI txwv enzymes los tsim recombinant plasmid pET28a-PEDVS, thiab cov fragments tshiab OMP16-PEDVS tau muab tso rau hauv pET28a thiab pVE5523 plasmids / PEDVS nrog EcoVRI thiab EcoVIR. txwv tsis pub enzymes los tsim cov plasmid recombinant pET28a-OMP16-PEDVS thiab pVE5523-OMP16-PEDVS, feem. Cov plasmids recombinant (pET28a-PEDVS, pET28a-OMP16-PEDVS, thiab pVE5523-OMP16-PEDVS) tau hloov mus ua BL21 (DE3) lossis Lactobacillus casei ATCC393 los ntawm kev hloov pauv lossis electroporation raws li daim ntawv qhia [21].
Kev tsom xam ntawm cov protein qhia los ntawm Western blot
Cov protein qhia ntawm BL21-pET28a-PEDVS, BL21- pET28a-OMP16-PEDVS, thiab L. casei-pVE5523-OMP16- PEDVS hom yog kuaj pom raws li txoj kev qhia nrog qee qhov kev hloov kho [21–23]. Cov recombinant hom BL21-pET28a-PEDVS thiab BL21-pET28a OMP16-PEDVS tau coj mus kuaj hauv LB broth thiab IPTG tau siv los sau rau pET28a-PEDVS thiab pET28a-OMP{{22} } PEVS proteins. Lub vector dawb paug tau siv los ua kev tswj tsis zoo. Tom qab lub cell lysis thiab centrifugation, lub supernatant thiab sediment tau sau. Tom qab ntawd, lub hom phiaj cov protein tau huv si siv cov Nickel affinity chromatography kem raws li kev tshawb fawb yav dhau los [24]. Lub caij no, cov kab lis kev cai supernatant ntawm recombinant L. casei pVE5523-OMP16-PEDVS hom kuj tau sau los ntawm centrifugation ntawm 9000 × g rau 10 min ntawm 4 degree . Tom qab ntawd, cov qauv nrog sodium dodecyl sulfate (SDS) loading tsis tau boiled 10 mins. Cov proteins tau muab cais los ntawm 12% sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) thiab tom qab ntawd xa mus rau hauv PVDF daim nyias nyias (Millipore, Mississauga, ON, Canada). Mem branes raug thaiv nrog 5% skimmed mis nyuj rau 2 teev ntawm 37 degree thiab tom qab ntawd incubated nrog murine monoclonal anti-lub cev ntawm S protein thaum hmo ntuj ntawm 4 degree thiab HRP conjugated tshis anti-nas IgG (ABclonal, Wuhan, Suav teb) rau 2 teev ntawm 37 degree. Cov kab protein tau pom pom siv Clarity ™ Western ECL Blotting Substrate (Bio-Rad, Hercules, CA, USA).
Kev txhaj tshuaj tiv thaiv thiab sau cov qauv
Cov tshuaj tiv thaiv kab mob ntawm cov tshuaj tiv thaiv Lactobacillus casei recombinant tau raug tshuaj xyuas los ntawm cov poj niam muaj hnub nyoog rau lub lis piam tshwj xeeb tsis muaj kab mob (SPF) BALB/c nas [10, 18], uas tau yuav los ntawm Shandong Agricultural University tsiaj chaw. Tag nrho ntawm 50 nas tau muab faib ua 5 pawg nrog 10 nas nyob hauv ib pab. Cov nas tau txhaj tshuaj tiv thaiv kab mob Lactobacillus casei recombinant thiab purified protein (pET28a-PEDVS, pET28a OMP16-PEDVS, pET28a-OMP16-PEDVS+Freund's ua tiav adjuvant), feem. Txoj kev txhaj tshuaj tiv thaiv kab mob tau ua raws li Table 2 thiab txhaj tshuaj tiv thaiv kab mob tom qab 13 hnub. Rau txoj kev tshawb fawb serological, cov ntshav tau sau rau ntawm 0, 14, thiab 28 hnub tom qab txhaj tshuaj tiv thaiv kab mob (dpi) ntawm tus Tsov tus tw leeg thiab khaws cia ntawm -20 degree kom txog thaum siv. Cov quav tau sau 1 hnub ua ntej txhaj tshuaj tiv thaiv thiab txhua lub sijhawm txhawb nqa. Rau kev tshawb nrhiav IgA, cov quav tau diluted (w / v) nrog 0.05 M sodium EDTA ntawm 1: 4 ratios tsuas yog tom qab sau thiab incubated rau 14 h ntawm 4 degree tom qab sib tov kom zoo. Lub supernatant tau sau los ntawm 12, 000 × g centrifugation thiab khaws cia ntawm − 20 degree kom txog thaum siv. 28 dpi; peb nas los ntawm txhua pab pawg tau muab txi thiab cov hnyuv tau ua tiav rau kev kuaj pom IgA raws li tus sau tau piav qhia yav dhau los [10, 18, 23].
Kev txiav txim siab ntawm cov tshuaj tiv thaiv kab mob
Cov theem ntawm IgG hauv sera thiab IgA hauv cov quav tau ntsuas los ntawm ELISA txoj kev nrog qee qhov kev hloov kho [18, 23]. Cov txheej txheem tau ua raws li hauv qab no: Polystyrene microtiter daim hlau raug coated thaum hmo ntuj ntawm 4 degree nrog 100 μL 10 ug/mL PEDVS protein, OMP16-PEDVS protein, los yog 100 μL recombinant L. casei-OMP16-PEDVS strain . Tom qab thaiv nrog 5% skimmed mis nyuj, cov qauv sau tau serially diluted hauv PBS, ntxiv rau hauv triplicate, thiab incubated ntawm 37 degree rau 1 h. Tom qab ntawd, HRP-conjugated tshis anti-nas IgG lossis IgA antibody (Invi trogen, USA) tau ntxiv rau txhua qhov dej (1: 5000) thiab incubated rau 1 h ntawm 37 degree. Cov phaj polystyrene microtiter tau ntxuav 5 zaug thaum txhua kauj ruam. Thaum kawg, 100 μL ntawm TMB substrate (tetramethylbenzidine thiab H2O2) tau ntxiv rau txhua qhov dej thiab 50 μL ntawm cov tshuaj nres tau ntxiv tom qab 10 mins. Qhov OD qhov tseem ceeb ntawm 630 nm tau ntsuas los ntawm kev nyeem ntawv multimode phaj (EnVision).
Kev kuaj kab mob neutralization
Lub neutralizing antibody titers ntawm PEDV hauv sera tau tshuaj xyuas raws li cov txheej txheem nrog qee qhov kev hloov kho [25, 26]. Luv luv, lub murine serum tau kub-tsis muaj zog (56 degree rau 30 min) thiab ces serially ob-fold diluted nyob rau hauv 96 zoo daim hlau (Corning, USA) nrog triplicates ntawm txhua tus qauv. Tom qab ntawd, qhov sib npaug ntawm 200 TCID50 / 50 μL PEDV hom tau muab ntxiv rau 96 qhov zoo daim hlau thiab incubated rau 1 h ntawm 37 degree. Cov sib tov tau ntxiv rau tshiab 96 zoo daim hlau coated nrog Vero cell monolayers thiab incubated rau 1 h ntawm 37 degree. Cells raug ntxuav thiab incubated nyob rau hauv lub txij nkawm nruab nrab ntawm 37 degree nyob rau hauv 5% CO2. Tom qab 2 hnub, cov nyhuv cytopathic (CPE) tau pom los ntawm kev siv lub tshuab ntsuas inverted, thiab qhov nruab nrab sizing concentration tau txhais tias yog qhov qis tshaj ntawm cov tshuaj tiv thaiv hauv cov ntshav.
Table 2 Qhov kev tiv thaiv kab mob ntawm cov nas BALB/c
Cytokine nrhiav pom
Txhawm rau txheeb xyuas qhov tso tawm ntawm cytokines, supernatants tau txais los ntawm cov nas kuaj (0, 14, thiab 28 hnub). Qib ntawm secreted IL-4, IL-10, thiab IFN- tau txiav txim siab siv cov khoom siv ELISA cov khoom lag luam (Elabscience Biotechnology Co., Ltd., Wuhan) raws li tus manu facturer cov lus pom zoo, feem. Cytokine tau txheeb xyuas los ntawm cov txheej txheem sib txawv uas tau npaj los ntawm cov txheej txheem reagents muab los ntawm cov khoom siv raws li cov khoom siv thiab cov nqi kho qhov muag (OD) tau kuaj pom ntawm 450 nm los ntawm txhua lub phaj siv lub phaj nyeem ntau hom (EnVision) [23, 27].
Kev txheeb cais
Tag nrho cov ntaub ntawv tau txais los ntawm tsawg kawg peb qhov kev sim ywj pheej, thiab cov txiaj ntsig tau nthuav tawm raws li qhov txhais tau tias ± tus qauv sib txawv (SD). Kev txheeb xyuas txheeb cais tau ua tiav siv ob-tailed t-tests thiab ib-txoj kev tsom xam hauv Graph Pad Prism 7.0 (GraphPad Software Inc., USA). Qhov sib txawv tseem ceeb tau txhais tias yog ∗ p < 0.05, thiab ntau qhov sib txawv ntawm qhov tseem ceeb tau raug xaiv raws li ∗∗ p < 0.01, ∗∗∗ thiab p < 0.001, ib.
Cov txiaj ntsig
Kev txheeb xyuas ntawm recombinant Lactobacillus casei thiab BL21 hom
Txhawm rau txheeb xyuas cov plasmids uas tau tsim ua, cov lus qhia rov ua dua plasmids pET28a-PEDVS, pET28a-OMP16-PEDVS, thiab pVE5523-OMP16-PEDVS tau zom nrog NcoI/XhoI txwv enzymes, thiab cov enzyme digestion tshwm sim nyob rau hauv daim duab 1. Qhov luaj li cas ntawm lub hom phiaj bands nyob rau hauv electrophoretograms yog tib yam li cov kev xav tau thiab sequencing tau qhia tias cov recombinant qhia plasmids qhia tsis muaj kev hloov. Cov txiaj ntsig no tau qhia txog kev ua tiav ntawm pET28a-PEDVS, pET28a OMP16-PEDVS, thiab pVE5523-OMP16-PEDVS recombinant plasmids. Cov txiaj ntsig ntawm sab hnub poob blot tau pom tias cov proteins sib xyaw pET28a-OMP16-PEDVS thiab pET28a-PEDVS tau qhia nyob rau hauv supernatant ntawm BL21-pET28a OMP16-PEDVS thiab BL21- pET28a-PEDVS hom kab mob, feem. Cov pVE5523-OMP16-PEDVS cov protein kuj tau txheeb xyuas hauv cov kab lis kev cai supernatant ntawm L. casei-pVE5523- OMP16-PEDVS hom. Cov kab tshwj xeeb los ntawm daim duab 2 tau pom tias cov proteins sib xyaw pET28a-OMP16- PEDVS, pVE5523-OMP16-PEDVS, thiab pET28a-PEDVS tau tag nrho cov qoob loo tiav lawm (Fig. 2).
Fig. 1 Cov enzyme digestion tshwm sim ntawm recombinant plasmids. A: Cov enzyme digestion tshwm sim ntawm pVE5523-OMP16-PEDVS plasmid. M: D8000 DNA Ladder Marker; 1: pVE5523-OMP16-PEDVS plasmid. B: Cov enzyme digestion tshwm sim ntawm pet28a-OMP16-PEDVS thiab pET28a-PEDVS plasmid. M: D8000 DNA Ladder Marker; 1: pet28a-OMP16-PEDVS plasmid; 2: pET28a-PEDVS plasmid
Cov qib IgG cov tshuaj tiv thaiv kab mob hauv cov ntshav ntawm cov nas txhaj tshuaj nrog cov tshuaj tiv thaiv neeg sib tw
Txhawm rau ntsuas qhov kev tiv thaiv kab mob tshwj xeeb ntawm cov neeg sib tw tshuaj tiv thaiv, BALB / c nas raug xaiv thiab muab faib ua 5 pawg. Tom qab ntawd, qib ntawm IgG hauv cov ntshav thiab IgA hauv cov quav tau ntsuas nrog cov khoom siv ELISA cov khoom lag luam. Cov txiaj ntsig tau qhia tias tsis muaj qhov sib txawv loj ntawm IgG qib ntawm cov pab pawg txhaj tshuaj thiab yuav luag tsis muaj cov tshuaj tiv thaiv IgG hauv txhua tus nas ua ntej txhaj tshuaj. Txawm li cas los xij, qhov sib txawv tseem ceeb tau pom tom qab tom qab txhaj tshuaj thawj zaug thiab qib IgG tshuaj tiv thaiv kab mob hauv cov ntshav ntawm 28 hnub tau siab dua li ntawm 14 hnub. Ntawm 5 pawg nas, cov nas txhaj tshuaj tiv thaiv kab mob L. casei-OMP16- PEDVS thiab BL21-OMP16-PEDVS-F pom tias muaj kev tiv thaiv kab mob zoo sib xws thiab siab tshaj plaws. Yog li, L. casei-OMP16- PEDVS thiab BL21-OMP16-PEDVS-F tuaj yeem tsim cov tshuaj tiv thaiv kab mob siab tshaj plaws, ua raws li BL21-OMP16- PEDVS thiab BL21-PEDVS (Fig. 3).
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Cov qib IgA cov tshuaj tiv thaiv kab mob hauv cov quav ntawm cov nas txhaj tshuaj nrog cov tshuaj tiv thaiv neeg sib tw
Txhawm rau ntsuas qhov tshwj xeeb immunogenicity ntawm cov neeg siv tshuaj tiv thaiv tsim tawm, qib ntawm IgA cov tshuaj tiv thaiv kab mob hauv cov quav ntawm cov nas kuj tau soj ntsuam. Cov txiaj ntsig tau pom tias tsis muaj tshuaj tiv thaiv tshwj xeeb PEDVS IgA cov tshuaj tiv thaiv muaj nyob ua ntej txhaj tshuaj. Txawm li cas los xij, ntau cov tshuaj tiv thaiv IgA hauv cov quav ntawm L. casei-OMP16-PEDVS txhaj tshuaj nas BL21-OMP16-PEDVS-F, BL21-OMP{{7} }PEDVS, thiab BL21-PEDVS pab pawg nas ntawm 14 hnub tom qab txhaj tshuaj. Thaum 28 hnub tom qab txhaj tshuaj, IgA cov tshuaj tiv thaiv kab mob ntawm L. casei-OMP16-PEDVS txhaj tshuaj nas mus txog qhov siab tshaj plaws. Lub caij no, IgA cov tshuaj tiv thaiv kab mob hauv lwm pab pawg tsis tau nthuav tawm qhov pom tseeb nce. Yog li ntawd, cov tshuaj tiv thaiv tus neeg sib tw L. casei OMP16-PEDVS tuaj yeem tsim cov tshuaj tiv thaiv kab mob ntau dua hauv cov nas uas tau txhaj tshuaj piv nrog BL21- OMP16-PEDVS-F, BL21-OMP{ {18}PEDVS, thiab BL21- PEDVS txhaj tshuaj nas (Daim duab 4).
Fig. 2 Kev txheeb xyuas ntawm cov proteins uas rov ua dua tshiab. M: protein marker; 1: daim ntawv secretory protein recombinant L. casei pVE5523-OMP16-PEDVS hom; 2: purified protein los ntawm BL21- pET28a-OMP16-PEDVS hom; 3: purified protein los ntawm BL21-pET28a-PEDVS hom
Lub neutralizing antibody qib ntawm cov ntshav hauv cov nas txhaj tshuaj
Txhawm rau ntsuas qhov kev tiv thaiv ntawm cov tshuaj tiv thaiv tus neeg sib tw ntawm L. casei-OMP16-PEDVS, BL21-OMP16-PEDVS-F, BL21- OMP16-PEDVS , thiab BL21-PEDVS hauv cov nas, cov tshuaj tiv thaiv tsis zoo tau raug ntsuas. Cov txiaj ntsig tau pom tias tsis muaj cov tshuaj tiv thaiv kab mob neutralizing ua ntej txhaj tshuaj. Neutralizing antibody tau kuaj pom ntawm 14 hnub tom qab txhaj tshuaj thiab nws nce ntxiv ntawm 14 hnub tom qab txhaj tshuaj tiv thaiv kab mob. Cov tshuaj tiv thaiv kab mob hauv cov nas uas tau txais L. casei-OMP16-PEDVS muaj qhov muaj zog tiv thaiv PEDV tsis ua haujlwm ntau dua li cov nas hauv qhov ncauj uas siv nrog BL21-OMP16-PEDVS-F, BL 21-OMP16-PEDVS, thiab BL21-PEDVS. Yog li, cov tshuaj tiv thaiv tus neeg sib tw L. casei OMP16-PEDVS tuaj yeem txhawb nqa qib siab tshaj plaws ntawm cov tshuaj tiv thaiv kab mob, ua raws li BL21-OMP16-PEDVS-F, BL21- OMP{{ 24}PEDVS, thiab BL21-PEDVS (Fig. 5).
Qib Cytokine
Txhawm rau sib piv cov kab mob tiv thaiv kab mob ntawm tes ntawm L. casei-OMP16-PEDVS, BL21-OMP16-PEDVS-F, BL21- OMP16-PEDVS, thiab BL21-PEDVS txhaj tshuaj tiv thaiv nas, IL- 4, IL-10, thiab IFN- tau txiav txim siab, feem. Cov txiaj ntsig tau pom tias qib ntawm cytokines IL-4, IL-10, thiab IFN- nyob rau hauv sera ntawm nas tau qis heev thiab tsis muaj qhov sib txawv tseem ceeb ua ntej txhaj tshuaj. Txawm li cas los xij, kev hloov pauv zoo sib xws tau pom hauv cov txiaj ntsig ntawm IL-4, IL-10, thiab IFN- . Thaum 14 hnub tom qab txhaj tshuaj, theem ntawm IL-4, IL-10, thiab IFN- hauv L. casei-OMP16-PEDVS cov nas txhaj tshuaj tau siab dua BL21- OMP16-PEDVS F, BL21-OMP16-PEDVS, thiab BL21-PEDVS txhaj tshuaj nas. Thaum 14 hnub tom qab txhaj tshuaj tiv thaiv kab mob, qhov siab dua IL-4, IL-10, thiab IFN- theem hauv L. casei-OMP16- PEDVS txhaj tshuaj nas tau kuaj pom piv nrog rau lwm yam. peb pawg. Yog li ntawd, tus neeg sib tw tshuaj tiv thaiv L. casei-OMP16-PEDVS tuaj yeem txhawb nqa qhov siab tshaj IL-4, IL-10, thiab IFN- theem, ua raws li BL21-OMP{{ 40}} PEDVS-F, BL21-OMP16-PEDVS, thiab BL21-PEDVS (Fig. 6).
Fig. 3 Cov tshuaj tiv thaiv IgG qib ntawm cov tshuaj tiv thaiv neeg sib tw hauv cov tshuaj tiv thaiv kab mob nas. Cov ntshav tau sau rau hnub 0, 14, thiab 28 hnub ua ntej lossis tom qab txhaj tshuaj tiv thaiv los ntawm cov khoom siv ELISA, thiab ntsuas ntawm qhov nqus ntawm 450 nm. Bars sawv cev qhov nruab nrab ± tus qauv sib txawv ntawm peb qhov kev sim ywj pheej. * p < 0.
Kev sib tham
Ib qho kev sib kis loj ntawm PED tshwm sim los ntawm PEDV qhov sib txawv tshwm sim thaum Lub Kaum Hli 2010, uas tau ua rau muaj kev poob nyiaj txiag loj heev hauv Suav teb thiab thoob plaws ntiaj teb [1]. Txawm li cas los xij, cov tshuaj tiv thaiv ib txwm yog txhua yam tsim los ntawm CV777 classical strain uas tsis tuaj yeem muab kev tiv thaiv txaus rau PEDV variant [4]. Txhawm rau tswj kev sib kis ntawm PEDV thiab txo qis kev lag luam, cov tshuaj tiv thaiv tshiab ntawm PEDV hom kab sib txawv kuj tau tsim. Tam sim no, PEDV cov tshuaj tiv thaiv tsis muaj zog thiab cov tshuaj tiv thaiv qis dua ntawm PEDV cov kab sib txawv yog txhua tus tau pom zoo los ntawm tsoomfwv Suav thiab muaj txhua yam pov thawj muaj kev cia siab rau kev tswj hwm PED. Tab sis qhov tsis xws luag kuj muaj nyob rau hauv ob hom tshuaj tiv thaiv tshiab. PEDV kis kab mob swine los ntawm txoj hnyuv thiab muaj cov hnyuv tropism. Yog li ntawd, Mucosal tiv thaiv zoo dua li kev tiv thaiv kab mob hauv kev tiv thaiv PEDV nkag mus rau hauv cov hnyuv epithelial hlwb, thiab cov tshuaj tiv thaiv yuav tsum muaj kev tiv thaiv mucosal zoo hauv txoj hnyuv. Yog li, Hauv txoj kev tshawb no, peb tsim ib hom tshuaj tiv thaiv tshiab uas tuaj yeem ua kom muaj zog tiv thaiv PEDV tshwj xeeb IgG thiab IgA cov tshuaj tiv thaiv. Lactobacillus casei muaj peev xwm tiv thaiv kab mob-modulatory zog ua lub tsheb xa tshuaj tiv thaiv thiab kev qhia txog bioactive compounds ntawm phab ntsa ntawm tes ntawm cov kab mob no tuaj yeem txhawb kev tiv thaiv kab mob tsim nyog [28, 29]. Nws yog dav siv los nthuav qhia ntau yam heterol ogous antigens ntawm tib neeg papillomavirus, Streptococcus pneumonia, thiab Escherichia coli ua cov tshuaj tiv thaiv hauv cov qauv tsiaj, uas txhua tus tau pom zoo heev immunogenicity [7- 9]. Piv nrog rau cov tshuaj tiv thaiv tsis muaj zog thiab cov tshuaj tiv thaiv qis qis, Lactobacillus casei vector tshuaj tiv thaiv tuaj yeem txhawb nqa IgA ntau dua thiab tiv thaiv kab mob hauv lub cev. Cov kev tshawb fawb tau pom tias IgA thawj kab kev tiv thaiv hauv cov hnyuv yuav zoo dua li IgG hauv kev tiv thaiv piglets los ntawm PEDV kab mob [10, 30]. Yog li ntawd, nws tau cog lus tias yuav tsim ib hom tshuaj tiv thaiv Lactobacillus casei vector ntawm PED. Raws li cov lus ceeb toom, S protein ntawm PEDV tuaj yeem muab faib ua S1 (1–735 amino acid) thiab S2 (736-kawg amino acid) domains [13], thiab S1 protein suav nrog thaj tsam receptor-binding thiab Lub ntsiab neutralizing epitopes [14] Lub core neutralizing epitope (COE) tuaj yeem ua rau muaj zog neutralizing antibodies tiv thaiv PEDV [31, 32]. Ua ke nrog kev tshuaj ntsuam antigenicity, ib feem ntawm S1 gene (1477-2124 bp) raug xaiv los tsim cov plasmid recombinant. Cov khoom me me uas tau xaiv tau ua pov thawj tias tsis yog tsuas yog muaj lub cev tiv thaiv kab mob zoo xwb tab sis kuj tseem ua rau muaj kev zais cia ntawm Lactobacillus casei. Ntawm qhov tod tes, Pasquevich pom tias Brucella abortus txheej membrane protein 16 tuaj yeem ua rau cov hlwb dendritic hauv vivo, ua rau muaj kev tiv thaiv kab mob th1, thiab yog ib qho kev cog lus rau tus kheej-adjuvanted tshuaj tiv thaiv kab mob thiab qhov ncauj tau txais brucellosis [15]. Cov kev tshawb fawb zoo sib xws uas unlipidated txheej membrane protein omp16 los ntawm Brucella spp. Raws li qhov ntswg adjuvant tuaj yeem ua rau lub cev tiv thaiv kab mob th1 thiab hloov kho qhov kev tsis haum th2 rau nyuj cov kua mis protein kuj tau ua pov thawj [16]. Yog li ntawd, ib feem ntawm omp16 noob tau raug xaiv los tsim cov plasmid recombinant los txhim kho kev tiv thaiv kab mob hauv peb txoj kev tshawb fawb. Txhawm rau paub seb qhov tshiab Lactobacillus casei pom zoo txhaj tshuaj tiv thaiv tuaj yeem ua rau cov lus teb rau lub cev tsis muaj zog, ntsuas IgG, IgA, thiab cov tshuaj tiv thaiv tsis zoo. Cov tshuaj tiv thaiv IgG ntawm L. casei-OMP16- PEDVS cov tshuaj tiv thaiv kab mob sib xyaw ua ke tsis muaj qhov sib txawv loj nrog BL21-OMP16-PEDVS-F recombinant txhaj tshuaj tiv thaiv nas. Txawm li cas los xij, qib IgA thiab neutralizing antibody siab dua li ntawm BL21-OMP16-PEDVS-F, BL21-OMP16-PEDV S, thiab BL{{47 }}PEDVS cov tshuaj tiv thaiv kab mob sib xyaw ua ke-imized nas. Cov txiaj ntsig tau pom tias L. casei-OMP16-PEDVS tuaj yeem ua rau muaj zog tiv thaiv kab mob, tshwj xeeb tshaj yog qib IgA tshuaj tiv thaiv. Cov kev tshawb fawb tau pom tias IgA yog thawj kab ntawm kev tiv thaiv hauv txoj hnyuv thiab yuav zoo dua li IgG hauv kev tiv thaiv piglets los ntawm PEDV kab mob [30]. Cov kev tshawb fawb kuj tau txheeb xyuas qhov tshwm sim tias L. casei-OMP16-PEDVS cov tshuaj tiv thaiv rov ua dua tuaj yeem muab kev tiv thaiv kab mob zoo dua rau PEDV. Txhawm rau tshawb xyuas hom kev tiv thaiv kab mob tiv thaiv kab mob los ntawm recombinant L. casei-OMP16-PEDVS, qib IL-4, IL-10, thiab IFN- tau tshawb pom los ntsuas qhov kev ua ntawm T lymphocytes. Raws li tsab ntawv ceeb toom, IFN- ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob ntawm tes los ntawm cov kab mob nkag mus rau hauv lub cev, IL-4 plays lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob humoral thiab txhawb kev tiv thaiv kab mob thiab kev tiv thaiv mucosal [33], IL -10 plays lub luag haujlwm tseem ceeb hauv kev tawm tsam cov kab mob mucosal microbial thiab tswj cov mucosal barrier kev ncaj ncees nyob rau hauv txoj hnyuv [34]. Lub caij no, cov txiaj ntsig ntawm kev tshawb nrhiav cytokine tau pom tias cov nas txhaj tshuaj nrog L. casei-OMP16-PEDVS cov tshuaj tiv thaiv kab mob tuaj yeem ua rau muaj zog dua ntawm IL-4, IL-10, thiab IFN- , uas txhawb nqa cov txiaj ntsig uas L. casei-OMP16-PEDVS cov tshuaj tiv thaiv sib xyaw ua ke tuaj yeem ua rau muaj zog tiv thaiv kab mob tiv thaiv kab mob, qib IgA tshuaj tiv thaiv kab mob, thiab cov tshuaj tiv thaiv kab mob ntawm tes, raws li.
Fig. 5 Qhov nruab nrab cov tshuaj tiv thaiv kab mob ntawm cov tshuaj tiv thaiv tus neeg sib tw hauv cov tshuaj tiv thaiv kab mob nas. Cov ntshav tau sau rau hnub 0, 14, thiab 28 hnub ua ntej lossis tom qab txhaj tshuaj tiv thaiv thiab tshuaj xyuas los ntawm kev kuaj tsis zoo. Bars sawv cev qhov nruab nrab ± tus qauv sib txawv ntawm peb qhov kev sim ywj pheej. * p < 0 . txawv
Fig. 6 Kev txheeb xyuas cov qib cytokine los ntawm cov ntshav ntawm cov nas txhaj tshuaj. Cov ntshav tau sau rau hnub 0, 14, thiab 28 hnub ua ntej lossis tom qab txhaj tshuaj thiab tshuaj xyuas los ntawm cov khoom siv ELISA cov khoom lag luam. Tus nqi absorbance tau ntsuas ntawm qhov nqus ntawm 450 nm rau IL-4 (a), IL-10 (b), thiab IFN- (c), feem. Bars sawv cev qhov nruab nrab ± tus qauv sib txawv ntawm peb qhov kev sim ywj pheej. * p < {{10}}.05, ** p < 0.01, thiab **** p < 0.0001 sawv cev nce qib ntawm qhov sib txawv tseem ceeb, raws li
Xaus
Hauv cov ntsiab lus, L. casei-OMP16-PEDVS, BL21-OMP16- PEDVS-F, BL21-OMP16-PEDVS, thiab BL{{7 }}PEDVS cov tshuaj tiv thaiv tus neeg sib tw tau tsim nyob rau hauv txoj kev tshawb no. Lub caij no, cov tshuaj tiv thaiv kab mob humoral thiab cellular immune teb qib ntawm cov tshuaj tiv thaiv tus neeg sib tw hauv nas tau raug soj ntsuam. Cov txiaj ntsig tau pom tias cov nas txhaj tshuaj nrog L. casei-OMP16-PEDVS tuaj yeem tsim cov qib siab dua ntawm IgG, IgA, neutralizing antibody, IL-4, IL- 10, thiab INF- piv nrog cov nas txhaj tshuaj nrog BL21-OMP16-PEDVS-F, BL21-OMP16-PEDVS, thiab BL21-PEDVS. Yog li ntawd, cov tshuaj tiv thaiv kab mob sib xyaw ua ke L. casei OMP16-PEDVS tus neeg sib tw tshuaj tiv thaiv tuaj yeem tsim lub hauv paus rau kev txhim kho kev nyab xeeb, zoo, thiab yooj yim siv tshuaj tiv thaiv mucosal rau kev tiv thaiv kab mob PEDV.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Cov ntaub ntawv
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