陳祎, 宋婷玉, 李金海, 肖永樂(lè), 曾光志, 萬(wàn)小平,楊璐一, 方鵬飛*, 王澤洲, 高榮*
1. 四川大學(xué)生命科學(xué)學(xué)院,生物資源與生態(tài)環(huán)境教育部重點(diǎn)實(shí)驗(yàn)室,動(dòng)物疾病防控和食品安全四川省重點(diǎn)實(shí)驗(yàn)室,成都610065;2. 四川省華派生物制藥有限公司,成都610026; 3. 四川省動(dòng)物疫病預(yù)防控制中心,成都610035)
細(xì)胞因子因其安全性和高效性而成為一種理想的免疫佐劑,例如γ-干擾素(IFN-γ)(Wangetal.,2013)、白細(xì)胞介素2(IL-2)(Yangetal.,2010)、白細(xì)胞介素4(IL-4)(Zhangetal.,2007)和白細(xì)胞介素6(IL-6)(Lietal.,2011)。IL-2、IL-4和IL-6在細(xì)胞和體液免疫反應(yīng)中起重要作用。IL-2具有多種生物學(xué)功能,包括促進(jìn)T細(xì)胞增殖和增強(qiáng)NK細(xì)胞的細(xì)胞毒性,刺激活化的B淋巴細(xì)胞增殖,并誘導(dǎo)免疫球蛋白分泌等(Collins & Oldham,1993)。IL-4可影響體液免疫和細(xì)胞免疫應(yīng)答,如免疫球蛋白的產(chǎn)生、類別轉(zhuǎn)換和分泌(Pasquinietal.,1997)。IL-6具有促進(jìn)白細(xì)胞介素基因表達(dá)、B細(xì)胞分化、T細(xì)胞活化的作用(Kishimoto,2010)。
豬IL-2或IL-6與CpG免疫刺激序列的混合基因能有效提高動(dòng)物對(duì)病原菌的抵抗力(Yangetal.,2010;Huangetal.,2013);此外,豬IL-4/6融合基因與單獨(dú)的IL-4和IL-6相比,能誘導(dǎo)機(jī)體產(chǎn)生更強(qiáng)的免疫應(yīng)答;豬IL-4/6融合基因能加強(qiáng)大腸桿菌疫苗、豬肺炎支原體疫苗、豬繁殖與呼吸綜合征(PRRS)疫苗對(duì)機(jī)體的體液和細(xì)胞免疫,提高接種動(dòng)物的免疫保護(hù)性(Zhangetal.,2007;Zhangetal.,2012;Yangetal.,2013);豬IL-2、IL-4、IL-6的融合基因(IL-4/6-2)相比于IL-4/6和IL-2在小鼠Musmusculus上可產(chǎn)生更強(qiáng)的免疫協(xié)同調(diào)節(jié)作用(楊璐一等,2014)。
因此,本實(shí)驗(yàn)在前期研究的基礎(chǔ)上,為研制新型高性價(jià)比的PCV-2疫苗免疫調(diào)節(jié)劑,評(píng)估共表達(dá)IL-2和IL-4/6融合基因納米顆粒對(duì)仔豬生長(zhǎng)和PCV-2免疫應(yīng)答的作用。
質(zhì)粒:真核表達(dá)載體VR1020-豬融合白細(xì)胞介素4/6-2(VRIL-4/6-2),由本實(shí)驗(yàn)室構(gòu)建并保存;殼聚糖(CS):15 kD,脫乙酰度95%以上,購(gòu)自Sigma Aldrich;多聚磷酸鈉(TPP):購(gòu)自Sigma(USA);鱟試劑:購(gòu)自湛江A & C公司;疫苗:PCV-2滅活疫苗(ZJ/C株)(圓環(huán)康)和實(shí)驗(yàn)動(dòng)物:健康21日齡長(zhǎng)白、約克夏和杜洛克雜交仔豬均由四川省華派生物制藥有限公司提供。
1.2.1VRIL-4/6-2真核表達(dá)質(zhì)粒的大量制備及內(nèi)毒素檢測(cè)參照《分子克隆指南 第三版》(Josephetal.,2002)制備質(zhì)粒,溶解于TE緩沖液,質(zhì)粒濃度和純度使用紫外分光光度計(jì)檢測(cè);質(zhì)粒內(nèi)毒素含量用鱟試劑檢測(cè)。
1.2.2VRIL-4/6-2質(zhì)粒殼聚糖納米顆粒的制備離子交聯(lián)法(Bodmeieretal.,1989)制備VRIL-4/6-2質(zhì)粒殼聚糖納米顆粒。將殼聚糖溶解于1%的冰醋酸溶液(pH5.5),配制為2.4 mg·mL-1的溶液;ddH2O配制10 mg·mL-1的磷酸三苯酯(TPP)溶液;以上溶液均用0.22 μm微孔濾膜過(guò)濾除菌。將VRIL-4/6-2質(zhì)粒與適量TPP溶液混勻,55 ℃孵育20 min;殼聚糖與質(zhì)粒質(zhì)量比為30∶1,在50~55 ℃水浴磁力攪拌下將質(zhì)粒與TPP的預(yù)混液緩慢滴加至殼聚糖溶液中,混合均勻,恒溫孵育10 min備用,記作VRIL-4/6-2-CS。用Zetasizer3000HS/IHPL粒度儀檢測(cè)納米顆粒粒徑和電位。
1.2.3動(dòng)物實(shí)驗(yàn)10頭21日齡長(zhǎng)白、約克夏和杜洛克雜交仔豬,經(jīng)ELISA和熒光定量PCR(qPCR)檢測(cè)PCV-2、PRRS病毒、豬瘟病毒和支原體均為陰性。隨機(jī)分成實(shí)驗(yàn)組和對(duì)照組,每組5頭。實(shí)驗(yàn)組頸部肌肉注射2.5 mL VRIL-4/6-2-CS(0.5 mg·mL-1),對(duì)照組注射相同劑量生理鹽水;2組均頸部肌肉注射2.5 mL PCV-2疫苗。豬免疫接種前記為第0天,接種后第7、14、28天定時(shí)采集前腔靜脈血用于檢測(cè)免疫反應(yīng)。接種前及實(shí)驗(yàn)結(jié)束第28天時(shí)分別測(cè)量2組仔豬的體質(zhì)量,用于評(píng)價(jià)實(shí)驗(yàn)質(zhì)粒對(duì)仔豬生長(zhǎng)的影響。
觀察組48例中,痊愈15例,顯效20例,有效10例,無(wú)效3例,總有效率為93.75%;對(duì)照組48例中,痊愈11例,顯效15例,有效12例,無(wú)效10例,總有效率為79.16%,觀察組明顯優(yōu)于對(duì)照組,兩組效果比較,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。
1.2.4PCV-2特異性抗體的測(cè)定取500 μL抗凝血低速離心后收集血清,參照ELISA試劑盒說(shuō)明書操作,在酶標(biāo)儀上測(cè)定OD630值,檢測(cè)血清中PCV-Ab的含量,試劑盒購(gòu)自武漢科前生物公司。
1.2.5IgG1和IgG2a的測(cè)定取500 μL抗凝血低速離心后收集血清,參照ELISA 試劑盒說(shuō)明書操作,在酶標(biāo)儀上測(cè)定OD450值,檢測(cè)血清中IgG1和IgG2a的含量,試劑盒購(gòu)自成都敏鑫科生物科技公司。
1.2.6CD4+和CD8+T淋巴細(xì)胞的測(cè)定取100 μL新鮮抗凝血搖勻,加入2 μL anti-porcine CD3-SPRD、2 μL anti-porcine CD4-FITC、2 μL anti-porcine CD8-PE,震蕩混勻避光孵育30 min;加入600 μL紅細(xì)胞裂解液,避光裂解10 min,500 r·min-1離心5 min,去上清;加入1 mL 磷酸緩沖鹽溶液(PBS),輕輕懸浮細(xì)胞,500 r·min-1離心5 min,去上清;重復(fù)洗滌1次,細(xì)胞沉淀加入300 μL PBS混勻,懸浮細(xì)胞,上機(jī)檢測(cè)。
1.2.7熒光定量檢測(cè)基因表達(dá)水平100 μL抗凝血加入1 mL的RNAiso pius,充分裂解后提取細(xì)胞總RNA,用TransScript All-in-One First-Strand cDNA Synthesis SuperMix for Qpcr (One-Step gDNA Removal)反轉(zhuǎn)錄試劑盒合成cDNA。根據(jù)GenBank中的豬PPIA、TLR-2、TLR-7、IL-2、IL-4、IL-6、TNF-α、STAT-1、STAT-2、STAT-3基因的cDNA序列,分別設(shè)計(jì)合成其特異性擴(kuò)增引物(表1)。
以仔豬不同時(shí)期血液cDNA為模板,表1中設(shè)計(jì)的引物進(jìn)行擴(kuò)增。用Bio-Rad IQ5熒光定量PCR儀檢測(cè)不同基因相對(duì)表達(dá)的情況,15 μL體系,用SsoAdvanceTMUniversal SYBR Green Supermix進(jìn)行熒光定量分析。擴(kuò)增參數(shù)為:95 ℃ 預(yù)變性30 s;95 ℃變性 5 s,最佳退火溫度退火30 s,40個(gè)循環(huán)。熔解曲線參數(shù):65~95 ℃,每6 s上升0.5 ℃。PPIA為內(nèi)參基因,采用2-ΔΔCT法分析實(shí)時(shí)熒光PCR數(shù)據(jù),比較同一目的基因不同時(shí)期的相對(duì)表達(dá)水平差異。
1.2.8數(shù)據(jù)分析以上各數(shù)據(jù)用GraphPad Prism 6中的雙因素方差分析和單因素方差分析進(jìn)行差異比較,P<0.05表示差異有統(tǒng)計(jì)學(xué)意義。
表1 熒光定量PCR特異性引物Table 1 The primers for qPCR
實(shí)驗(yàn)開(kāi)始及結(jié)束時(shí)分別稱量每只仔豬的體質(zhì)量,分別計(jì)算每組仔豬體質(zhì)量的平均值和方差,仔豬體質(zhì)量變化見(jiàn)表2。結(jié)果顯示,實(shí)驗(yàn)組仔豬體質(zhì)量增加顯著高于對(duì)照組(P<0.05)。
表2 實(shí)驗(yàn)28天仔豬體質(zhì)量變化Table 2 Body mass change of piglets during 28 days
注 Note:*P<0.05
在接種第7天后,實(shí)驗(yàn)組和對(duì)照組中均可以檢測(cè)到PCV-2抗體,但是2組含量之間的差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)(圖1)。
圖1 仔豬血清中PCV-2抗體含量的變化Fig. 1 PCV-2 antibody titers in the serum of the experimental piglets
S/P=(S-N)/(P-N)=(樣品OD值-陰性對(duì)照OD值)/(陽(yáng)性對(duì)照OD值-陰性對(duì)照OD值), 當(dāng)S/P≥0.16時(shí), 認(rèn)為PCV-2抗體檢測(cè)為陽(yáng)性
S/P=(S-N)/(P-N)=(ODsample-ODnegative control)/(ODpositive control-ODnegative control), titer of PCV-2 antibody was considered positive whenS/P≥0.16
實(shí)驗(yàn)組仔豬血清中IgG1的含量顯著低于對(duì)照組(P<0.05),而IgG2a的含量顯著高于對(duì)照組(P<0.05)(圖2)。
流式細(xì)胞術(shù)檢測(cè)仔豬血液中CD4+和CD8+T淋巴細(xì)胞亞群的數(shù)量變化,實(shí)驗(yàn)組仔豬血液中的CD4+T和CD8+T淋巴細(xì)胞的數(shù)量與對(duì)照組相比均顯著增加(P<0.05)(圖3)。
2.5.1Toll-like受體基因表達(dá)定量分析實(shí)驗(yàn)組TLR-2基因的表達(dá)水平只在免疫后第14天顯著高于對(duì)照組(P<0.05),而實(shí)驗(yàn)組TLR-7基因的表達(dá)水平在免疫后第28天顯著高于對(duì)照組(P<0.05)(圖4)。
圖2 仔豬血清中IgG1和IgG2a含量的變化Fig. 2 The change of the levels of IgG1 and IgG2a in the serum of piglets
圖3 仔豬血液中 CD4+和CD8+T淋巴細(xì)胞數(shù)量的變化Fig. 3 CD4+ and CD8+ T cell numbers in the blood of experimental piglets
2.5.2細(xì)胞因子基因表達(dá)定量分析實(shí)驗(yàn)組仔豬血液中IL-2、IL-4、IL-6和TNF-α基因的表達(dá)水平在免疫后第28天均顯著高于對(duì)照組(P<0.05),其中,IL-4和IL-6基因表達(dá)水平分別在第7天和第14天顯著高于對(duì)照組(P<0.05)(圖5)。
2.5.3免疫信號(hào)傳導(dǎo)分子基因表達(dá)定量分析STAT-1基因表達(dá)水平在免疫后第7~14天均顯著高于對(duì)照組(P<0.05),STAT-2基因表達(dá)水平在免疫后第14~28天顯著高于對(duì)照組(P<0.05),STAT-3基因表達(dá)水平在免疫后第7、28天顯著高于對(duì)照組(P<0.05)(圖6)。
細(xì)胞因子是調(diào)節(jié)動(dòng)物免疫功能的關(guān)鍵分子。許多細(xì)胞因子被證實(shí)作為佐劑對(duì)體液或細(xì)胞免疫有增強(qiáng)作用(Pasquinietal.,1997;Kayamuroetal.,2010)。IL-2不僅支持T細(xì)胞和NK細(xì)胞的活化和增殖,還可以刺激和活化B淋巴細(xì)胞增殖和誘導(dǎo)免疫球蛋白分泌(Smith,1988;Collins & Oldham,1993)。IL-4可促進(jìn)體液免疫,增加特異性和非特異性殺傷功能(Erbetal.,1997;Paul,2015)。IL-6可調(diào)節(jié)B細(xì)胞活化、抗體產(chǎn)生和Th1型免疫反應(yīng)(Paul & Seder,1994;Kishimoto,2006)。有研究表明,豬IL-4/6、IL-2和IL-4/6-2融合基因均可安全增強(qiáng)動(dòng)物系統(tǒng)和全面的免疫(Yangetal.,2010,2013;楊璐一等,2014)?;谝郧暗难芯?,本研究第一次將豬IL-4/6-2融合基因應(yīng)用到豬上,來(lái)加強(qiáng)對(duì)PCV-2疫苗的免疫效果。
圖4 仔豬TLR基因表達(dá)水平的變化Fig. 4 The change of expression levels of TLR gene in the blood of experimental piglets
圖5 仔豬細(xì)胞因子基因表達(dá)水平的變化Fig. 5 The change of cytokine gene expression in the blood of experimental piglets
圖6 仔豬免疫信號(hào)傳導(dǎo)分子基因表達(dá)水平的變化Fig. 6 The change of the immune signal transduction molecules related genes in the blood of experimental piglets
本實(shí)驗(yàn)用殼聚糖將重組質(zhì)粒VRIL-4/6-2包裹,制備殼聚糖納米顆粒,與PCV-2疫苗同時(shí)對(duì)實(shí)驗(yàn)仔豬進(jìn)行肌肉注射免疫接種。實(shí)驗(yàn)所用重組質(zhì)粒通過(guò)2A短肽連接3種白細(xì)胞介素基因,構(gòu)建共表達(dá)質(zhì)粒。2A短肽具有的自剪切機(jī)制使連接前后的基因表達(dá)量相同(de Felipe & Ryan,2004;Szymczak & Vignali,2005)。裸露的基因不穩(wěn)定且半衰期短,轉(zhuǎn)染效率低;而殼聚糖包裹質(zhì)??尚纬尚〉膸д姾傻募{米顆粒,與裸露的質(zhì)粒相比,它可以更有效地?cái)y帶目的基因進(jìn)入細(xì)胞,還可以抑制其在生物體內(nèi)降解,被證實(shí)是一種有潛力的緩釋材料,并且還具有生物相容性(Chewetal.,2003;Cui & Mumper,2003),因此,殼聚糖作為基因載體的研究發(fā)展迅速(Buschmannetal.,2013;Fernándezetal.,2016)。Huang等(2005)通過(guò)熒光標(biāo)記研究殼聚糖-DNA復(fù)合粒子進(jìn)入細(xì)胞的過(guò)程,證實(shí)了殼聚糖-DNA復(fù)合粒子可以被細(xì)胞內(nèi)吞,繼而進(jìn)行后續(xù)基因轉(zhuǎn)染。之前的研究也表明,殼聚糖納米粒子在動(dòng)物身上用于基因傳遞幾乎沒(méi)有毒性和影響(Yangetal.,2010;Zhangetal.,2012),并且空白載體質(zhì)粒組與空白組沒(méi)有差異(Yangetal.,2010)。本次實(shí)驗(yàn)由于陰性動(dòng)物難于篩選獲得,數(shù)量有限,并未進(jìn)行空白載體質(zhì)粒組的實(shí)驗(yàn)。
在實(shí)驗(yàn)期間,實(shí)驗(yàn)組和對(duì)照組仔豬注射局部均未出現(xiàn)病變、損傷或其他系統(tǒng)性癥狀。實(shí)驗(yàn)開(kāi)始和結(jié)束時(shí)分別記錄了每只仔豬的體質(zhì)量,結(jié)果顯示,實(shí)驗(yàn)組仔豬的生長(zhǎng)速率顯著高于對(duì)照組。有研究顯示,IL-2、IL-4、IL-6、IL-10、TNF-α等細(xì)胞因子可在豬小腸中持續(xù)表達(dá),并且細(xì)胞因子除了直接的免疫調(diào)節(jié)作用,還可影響上皮細(xì)胞的生長(zhǎng)、內(nèi)環(huán)境穩(wěn)態(tài)和免疫細(xì)胞的運(yùn)輸(Oswald,2006;Devriendtetal.,2010)。推測(cè)VRIL-4/6-2質(zhì)粒對(duì)調(diào)節(jié)仔豬內(nèi)分泌、促進(jìn)物質(zhì)代謝有積極作用。
PCV-2特異性抗體和PCV-2中和抗體是體液免疫反應(yīng)的重要檢測(cè)指標(biāo)(Meertsetal.,2006;Fortetal.,2007)。研究結(jié)果表明,實(shí)驗(yàn)組和對(duì)照組仔豬血清中PCV-2抗體的含量差異無(wú)統(tǒng)計(jì)學(xué)意義,且實(shí)驗(yàn)組仔豬血清中IgG1的含量顯著低于對(duì)照組,這表明IL-4/6-2可能沒(méi)有明顯增強(qiáng)仔豬對(duì)PCV-2疫苗的體液免疫。此外,有研究顯示,誘導(dǎo)細(xì)胞免疫對(duì)于PCV-2的防控很有必要(Fortetal.,2009)。本研究結(jié)果表明,實(shí)驗(yàn)組的CD4+和CD8+T淋巴細(xì)胞數(shù)量顯著高于對(duì)照組,并且實(shí)驗(yàn)組的IL-2、TNF-α基因表達(dá)水平在第28天顯著高于對(duì)照組。IL-2、IFN-γ和TNF-α均由Th1細(xì)胞產(chǎn)生,Th1細(xì)胞可誘導(dǎo)巨噬細(xì)胞活化、遲發(fā)性超敏反應(yīng)和IgG2a的產(chǎn)生(Mosmann & Coffman,1989;Abbasetal.,1996)。同時(shí),本次實(shí)驗(yàn)發(fā)現(xiàn),接種質(zhì)粒的實(shí)驗(yàn)組比對(duì)照組產(chǎn)生了更多的IgG2a抗體,表明實(shí)驗(yàn)組仔豬產(chǎn)生了更強(qiáng)的Th1型免疫應(yīng)答,相比對(duì)照組,VRIL-4/6-2-CS提高了仔豬對(duì)PCV-2疫苗的細(xì)胞免疫應(yīng)答。
另外,實(shí)驗(yàn)組仔豬血液中IL-4、IL-6、TLR-2、TLR-7、STAT-1、STAT-2、STAT-3基因的表達(dá)水平在不同時(shí)間段內(nèi)也顯著高于對(duì)照組。TLRs是先天免疫系統(tǒng)的重要組成部分,Toll-like受體在先天免疫中識(shí)別不同病原菌的保守分子模式,并參與對(duì)病原體的特異性體液和細(xì)胞免疫反應(yīng)的激活(Kawai & Akira,2010)。STAT-1、STAT-2參與IFNs應(yīng)答反應(yīng),并在干擾素抗病毒應(yīng)答調(diào)節(jié)中起重要作用(Mitchell & John,2005;Steen & Gamero,2013);STAT-3可由多種細(xì)胞因子激活,例如IL-6、IL-12具有有效的抗炎作用,可調(diào)節(jié)細(xì)胞生長(zhǎng)、凋亡、炎性基因的轉(zhuǎn)錄等重要細(xì)胞過(guò)程(Egwuagu,2009);JAK激酶和STAT蛋白參與了許多細(xì)胞因子的信號(hào)轉(zhuǎn)導(dǎo),它在細(xì)胞因子介導(dǎo)的免疫反應(yīng)和調(diào)控中具有重要作用(Shuai & Liu,2003)。綜上,這些基因表達(dá)水平的提高表明VRIL-4/6-2-CS誘導(dǎo)了更加全面的免疫應(yīng)答,增強(qiáng)了仔豬的先天性和適應(yīng)性免疫應(yīng)答。
總之,本研究結(jié)果首次證實(shí)VRIL-4/6-2-CS可有效協(xié)同增強(qiáng)仔豬對(duì)PCV-2疫苗的免疫應(yīng)答。VRIL-4/6-2-CS是一種新型高性價(jià)比佐劑,可提高豬對(duì)抗PCV-2感染的免疫預(yù)防能力,具有重要應(yīng)用前景。
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