CN117886953A - 重组AaLS-IDR-1047免疫增强肽、制备方法及应用 - Google Patents
重组AaLS-IDR-1047免疫增强肽、制备方法及应用 Download PDFInfo
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Abstract
本发明于生物技术领域，公开了重组AaLS‑IDR‑1047免疫增强肽、制备方法及应用，所述的免疫增强肽为SEQ ID NO.2所示。将纯化后的免疫增强肽AaLS‑IDR‑1047作为免疫增强剂按一定剂量添加进伪狂犬病灭活疫苗中，增强机体对抗原产生的中和抗体水平，降低伪狂犬病毒在脑组织中的载毒量，提高疫苗的保护率。将AaLS‑IDR‑1047添加进倍比稀释抗原量的伪狂犬病疫苗中，可降低配制伪狂犬病疫苗的抗原量，同时降低疫苗对动物机体的局部炎症反应以及生长性能的影响，并在猪上验证了重组AaLS‑IDR‑1047免疫增强肽的免疫增强效果，有效增加了其抗体水平和细胞因子水平。
Description
技术领域
本发明属于生物技术领域，具体涉及重组AaLS-IDR-1047免疫增强肽、制备方法及应用。
背景技术
来源于超嗜热菌Aquifex aeolicus的Lumazine合酶(AaLS)由60个相同的亚基组成，具有球形和空心二十面体衣壳结构，具有显著的热稳定性，通常形成特定的多聚体结构，例如环形或笼形结构，能形成一种自组装蛋白质纳米颗粒(Kim et al 2022；Zhang etal 2001)。这些组装体的颗粒性质和重复的亚基组织能够作为抗原递呈的展示平台(Lamontagne et al 2022)，并产生高于单体蛋白支架产生的抗体水平(LadensteinMorgunova 2020)。
IDRs(Innate defense regulators)，也称为阳离子多肽或者天然防御调节肽，IDRs是宿主防御肽(HDPs)的合成类似物，筛选自牛溶菌肽(bovine bactenecin Bac2A)。人工合成的IDRs减少了对微生物产物的促炎反应，从而限制了潜在有害的炎症(Scott,M.G.et al 2007；Nijnik,A.et al 2010)。例如，在小鼠模型中，原型IDR-1对多重耐药菌的系统性感染提供预防和治疗保护，这种保护与增加趋化因子的产生、抑制有害的炎症细胞因子和感染部位巨噬细胞的增加有关(Scott,M.G.et al 2007)。IDR-1002通过增强趋化因子的产生以及PMN白细胞/中性粒细胞和单核/巨噬细胞在体内感染部位的募集来增加对细菌感染的保护(Nijnik,A.et al 2010)，IDR-HH2也显示出作为单剂量疫苗佐剂的潜力(Kindrachuk,J.et al 2009；Gracia,A.et al 2011；Brown,T.H.et al 2012)。IDR-1018是迄今为止最有效的细胞因子诱导剂(Wiecz orek,M.et al 2010)，在小鼠模型中显示出抗感染和抗炎活性，包括与标准一线抗疟药联合使用时治疗伯氏疟原虫ANKA脑型疟疾的疗效(Achtman,A.H.et al 2012)。IDR介导的细胞募集机制涉及局部趋化因子(Scott,M.G.etal 2007；Nijnik,A.et al 2010；Wieczorek,M.et al 2010；Le e,H.Y.et al 2008)的诱导和促进整合素介导的黏附(Madera,L.et al 2012)。
本发明通过基因工程的方法设计引物使AaLS和天然防御调节肽IDR(Innatedefense reg ulators)串联，对AaLS-IDR-1047进行表达。首次发现AaLS-IDR-1047具有免疫增强剂的效果，将纯化后的免疫增强肽AaLS-IDR-1047作为免疫增强剂按一定剂量添加进灭活PRV疫苗中，增强机体对抗原产生的中和抗体水平，降低PRV病毒在脑组织中的载毒量，提高保护率，同时降低疫苗对动物机体的局部炎症反应以及生长性能的影响，并在猪上验证了重组AaLS-IDR-1047免疫增强肽的免疫增强效果，有效增加了其抗体水平。
发明内容
本发明的目的在于提供了重组AaLS-IDR-1047免疫增强肽，所述免疫增强肽的氨基酸序列为SEQ ID NO.2所示。
本发明的的另一个目的在于提供了重组AaLS-IDR-1047免疫增强肽的微生物制备方法，方法简单，蛋白表达量高。
本发明的最后一个目的在于提供了重组AaLS-IDR-1047免疫增强肽作为免疫增强剂在伪狂犬病灭活疫苗中的应用。
为了达到上述目的，本发明采取以下技术措施：
AaLS-IDR-1047免疫增强肽，所述免疫增强肽为SEQ ID NO.2所示。利用化学合成或是微生物表达的方式，均能获得上述免疫增强肽。
编码SEQ ID NO.2所示蛋白的基因。
含有上述基因的表达载体。
表达含有SEQ ID NO.2所示蛋白的重组微生物。
AaLS-IDR-1047免疫增强肽的制备方法，包括：将编码SEQ ID NO.2所示蛋白的基因插入表达载体，转化大肠杆菌进行诱导表达。
以上所述的制备方法，优选的，编码SEQ ID NO.2所示蛋白的基因为SEQ ID NO.1所示。
本发明的保护范围还包括：
AaLS-IDR-1047免疫增强肽在制备疫苗免疫增强剂中的应用。
以上所述的应用中，优选的，所述的免疫增强剂的应用对象为小鼠和/或猪。
以上所述的应用中，优选的，所述的疫苗为猪伪狂犬灭活疫苗。
与现有技术相比，本发明具有以下优点：
1.本发明首次制备了AaLS-IDR-1047融合蛋白，并验证其具有显著免疫增强效果。
2.AaLS-IDR-1047免疫增强肽添加进入猪伪狂犬灭活疫苗能够显著提高免疫小鼠、猪的中和抗体效价，Th1型和Th2型细胞因子及肿瘤坏死因子分泌水平。
3.AaLS-IDR-1047免疫增强肽添加进伪狂犬灭活疫苗中可以降低达到足够血清中和抗体所需的伪狂犬抗原量，降低成本。
附图说明
图1为重组IDR-1047免疫增强肽的表达与纯化示意图；
A：泳道1：空白对照；泳道2：诱导全菌蛋白表达情况；泳道3：诱导上清中的IDR-1047蛋白，大小约40kDa；泳道4：纯化流穿液；泳道5：50MM咪唑洗脱液；泳道6：100MM咪唑洗脱液；泳道7：150MM咪唑洗脱液。
B：AaLS-IDR-1047纯化后蛋白质免疫印记结果，泳道1为未纯化AaLS-IDR-1047；泳道2为PBS；泳道3为纯化AaLS-IDR-1047。
图2为AaLS-IDR-1047免疫小鼠的血清中和抗体水平图像。
图3为AaLS-IDR-1047免疫小鼠的脑组织伪狂犬病毒绝对定量PCR标准曲线图像。
图4为倍比稀释伪狂犬抗原后AaLS-IDR-1047免疫小鼠的血清中和抗体水平图像。
图5为倍比稀释伪狂犬抗原后AaLS-IDR-1047免疫小鼠的脑组织免疫荧光图像，刻度尺为500um，其中A为Vac组；B为Vac+AaLS-IDR-1047组；C为1/2抗原Vac+AaLS-IDR-1047组；D为；1/3抗原Vac+AaLS-IDR-1047组；E为；1/4抗原Vac+AaLS-IDR-1047组；F为；1/5抗原Vac+AaLS-IDR-1047组。
图6为AaLS-IDR-1047免疫猪的血清中和抗体图像。
图7为AaLS-IDR-1047免疫猪21天后细胞因子水平示意图；
其中：A:Th1型(干扰素-γ)；B:Th2型(IL-6)；C：肿瘤坏死因子(TNF-α)。
具体实施方式
本发明所述技术方案，如未特别说明，均为本领域的常规方案；所述试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：
融合蛋白AaLS-IDR-1047的表达与纯化：
在申请CN116769053A的基础上，申请人发现，将IDR-1047(多肽序列为：VEQVFWRERHRI)与突变后的AaLS(SEQ ID NO.4所示，由SEQ ID NO.3所示基因编码)结合后，获得的融合蛋白AaLS-IDR-1047具有免疫增强剂的作用，所述的融合蛋白为SEQ ID NO.2所示，编码其的基因为SEQ ID NO.1所示。
本发明，采取大肠杆菌发酵的方式制备该融合蛋白，其余方式，例如人工合成，其他微生物发酵也可获得本发明的融合蛋白AaLS-IDR-1047。
具体方式如下：
(1)重组AaLS-IDR-1047免疫增强肽基因重叠延伸PCR扩增
根据大肠杆菌偏嗜密码子设计重组AaLS-IDR-1047免疫增强肽基因，并针对该融合肽基因设计引物F1：5’-TggaacaggtgttttggcgcgaacgccatcgcattGTGCGCCTGCGCTGGTGG-3’和F2：5’-GccaaaacacctgttccacCATCTTGTCGTCGTCGTCGG-3’，F3：5’-tggaacaggtgttttggcgcg aacgccatcgcattGGATCGGGCTCTCACCACC-3’，和F4：5’-gccaaaacacctgttccacGCCTCCTC CTGAGCCTCCA-3’，然后进行SOE-PCR扩增；将PCR扩增产物采用琼脂糖凝胶电泳鉴定后,切下目的条带进行回收,回收产物中片段大小为663bp的为重组AaLS-IDR-1047免疫增强肽基因。
(2)携带重组AaLS-IDR-1047免疫增强肽基因的工程菌的构建
将重组AaLS-IDR-1047免疫增强肽基因和质粒载体pET32a进行酶切连接，连接产物转化E.coli DH5α并提取质粒进行鉴定，片段大小为1140bp，正确质粒命名为pET32a-AaLS-IDR-1047；重组质粒pET32a-AaLS-IDR-1047转化进入感受态大肠杆菌BL21(DE3)(唯地生物，EC1002)中。筛选阳性克隆即为表达重组AaLS-IDR-1047免疫增强肽的工程菌。(3)重组AaLS-DR-1047免疫增强肽蛋白表达
将已验证的感受态大肠杆菌接种于含100mg/ml氨苄西林(Ampicillin)的Luria-Bertani(LB)1.0L培养基中37℃培养。在OD600 nm吸光密度为0.6～0.8的条件下，用400μMIPT G诱导细胞表达蛋白，30℃诱导12小时。收集细胞，PBS(pH＝7.4)重悬，高压破碎，离心(12000×g，30min)取上清。
(4)重组AaLS-IDR-1047免疫增强肽蛋白纯化
采用镍柱亲和层析纯化蛋白，即可获得单一的重组AaLS-IDR-1047融合肽。操作步骤如下：
将所有过柱的液体用0.22m的过滤器过滤；将纯化柱中的20·％乙醇流净，用10个柱体积的蒸留水洗去填料中的乙醇，再加入10个柱体积的洗脱缓冲液洗净填料，最后加入10个柱体积的结合缓冲液平衡填料，为蛋白结合提供合适的环境；加5mL样品到纯化柱中，收集穿流液，反复加样3-4次；纯化柱用10个柱体积的结合缓冲液穿流平衡填料；配置不同浓度咪唑洗脱液(0·mM、15mM、30mM、50mM、100mM、200·mM、250mM、300mM咪，pH-7.4)，采用逐步洗脱或线性梯度洗脱，洗完之后加入洗脱缓冲液洗柱子，清理一切蛋白洗脱，收集洗脱液；纯化柱的洗涤与回收:向纯化柱中加入20L洗脱缓冲液洗涤，然后加入20mL蒸馏水洗涤，再加入20·mL结合缓冲液平衡，最后加满20·％乙醇4·℃正立保存；每个浓度取40uL制样进行PAGE胶电泳(图1中A)，判断最佳洗脱目的蛋白浓度和杂蛋白浓度为150mM咪唑。
纯化后的目的蛋白可以进一步浓缩透析以获得单一高浓度重组AaLS-IDR-1047免疫增强肽，并采用Bradford蛋白质定量试剂盒(TIANGEN，北京，中国)对所得蛋白的浓度进行测定，经换算每1L培养基表达28.4mg重组AaLS-IDR-1047融合蛋白，SEQ ID NO.2所示。
蛋白质免疫印记结显示，AaLS-IDR-1047能够在上清中稳定表达(图1中A)以单一蛋白质带的形式在40kDa和43kDa之间迁移(图1中B)。
综上，免疫增强肽IDR-1047构建于AaLS蛋白骨架上，形成融合蛋白后，能够稳定通过大肠杆菌表达系统进行表达，并借助His标签进一步纯化，从而得到稳定单一的目的蛋白，除了构建Aals-IDR-1047，按照上述方法构建了Aals-IDR-1002(IDR-1002的序列为VQRWLIVWRIRK)、Aals-IDR-HH2(IDR-HH2的序列为VQLRIRVAVIRA)、Aals-IDR-1(IDR-1的序列为KSRIVPAIPVSLL)、Aals-IDR-1018(IDR-1018的序列为VRLIVAVRIWRR)等融合蛋白，共同检测是否具备免疫增强效果。
实施例2：
重组AaLS-IDR-1047免疫增强肽在伪狂犬病灭活疫苗中的免疫增强作用
(1)疫苗的制备
用上述表达纯化后的AaLS-IDR-1047等免疫增强肽与伪狂犬(PRV)灭活抗原(Vac)作为水相按照合适的体积比与油相201佐剂采用低剪切搅拌器1000转/每分钟乳化15分钟后，经检测剂型为水包油包水，性质稳定，黏度合适即为成品苗(AaLS-IDR-1047+Vac)，储存于4℃。
按照上述方法，制备了疫苗AaLS-IDR-1002+Vac、AaLS-IDR-HH2+Vac、AaLS-Aals-IDR-1+Vac和AaLS-Aals-IDR-1018+Vac。
按照多肽序列，直接化学合成IDR-1047(生工生物，上海)，按照上述方法制备疫苗ID R-1047+Vac。
(2)免疫增强肽免疫保护试验
免疫前对每只小鼠称重，免疫后每周称重。每组第一次免疫28日龄SPF小鼠，肌肉免疫，0.2ml/只。每组21d后第二次免疫，肌肉免疫，0.2ml/只。
实验分为八组：
PBS对照组：PBS作为水相与201佐剂乳化，使用时，0.2ml/只。
Vaccine组(伪狂犬病灭活疫苗免疫组)：该疫苗在使用时，疫苗中的伪狂犬病毒灭活抗原为2*107.0TCID50/ml。
IDR-1047+Vac组：该疫苗在使用时，疫苗中的IDR-1047为10μg/只，Vac(即伪狂犬灭活抗原)为2*107.0TCID50/ml。
AaLS-IDR-1047+Vac组：该疫苗在使用时，疫苗中的AaLS-IDR-1047为10μg/只，Vac(即伪狂犬灭活抗原)为2*107.0TCID50/ml。
AaLS-IDR-1002+Vac组：该疫苗在使用时，疫苗中的AaLS-IDR-1002为10μg/只，Vac(即伪狂犬灭活抗原)为2*107.0TCID50/ml。
AaLS-IDR-HH2+Vac组：该疫苗在使用时，疫苗中的AaLS-IDR-HH2为10μg/只，Vac(即伪狂犬灭活抗原)为2*107.0TCID50/ml。
AaLS-IDR-1+Vac组：该疫苗在使用时，疫苗中的AaLS-IDR-1为10μg/只，Vac(即伪狂犬灭活抗原)为2*107.0TCID50/ml。
AaLS-IDR-1018+Vac组：该疫苗在使用时，疫苗中的AaLS-IDR-1018为10μg/只，Vac(即伪狂犬灭活抗原)为2*107.0TCID50/ml。
一免后14d断尾采血分离血清，采用微量血清中和抗体实验检测伪狂犬抗体水平。二免后14d断尾采血分离血清，采用微量血清中和抗体实验(SMX毒株)检测伪狂犬抗体水平。二免采血后7d攻毒(伪狂犬SMX毒株，LD50＝19.5TCID50)20倍LD50 200ul/只，取脑组织，研磨后提取DNA，绝对荧光定量PCR检测脑组织伪狂犬病毒载毒量，绘制CT值与载毒量的标准曲线(图3)，计算伪狂犬病毒检出率与载毒量。
结果显示，添加AaLS-IDR-1047免疫增强肽的疫苗组血清中和抗体水平(图2)最高，显著高于伪狂犬疫苗对照组和其他融合蛋白组，并且脑组织中伪狂犬病毒的载毒量及检出率最低(表1)，其他融合蛋白如Aals-IDR-1002和Aals-IDR-HH2不仅没有增强免疫反应，甚至降低了抗体水平(图2)，其中Aals-IDR-1002组小鼠脑组织伪狂犬病毒检出率及载毒量相比于疫苗对照组更高；而Aals-IDR-1和Aals-IDR-1018与伪狂犬疫苗对照组产生的抗体水平类似，效果不如AaLS-IDR-1047。这些结果证实AaLS-IDR-1047在小鼠免疫中具有免疫增强活性。
表1小鼠第二次免疫14天后伪狂犬病灭活疫苗和不同浓度免疫增强剂组保护效果
| 分组 | 黏度/cP | 增重/g | 检出率 | 载毒量 |
| PBS | / | 14.52±1.22 | 9/10 | 104.5 |
| Vaccine | 42.0 | 15.11±1.24 | 7/10 | 104.1 |
| IDR-1047+Vac | 43.5 | 15.64±0.85 | 6/10 | 104.0 |
| AaLS-IDR-1047+Vac | 44.1 | 15.82±2.12 | 5/10 | 103.3 |
| Aals-IDR-1002+Vac | 42.5 | 14.88±1.12 | 8/10 | 104.2 |
| Aals-IDR-HH2+Vac | 43.6 | 15.09±1.32 | 7/10 | 104.0 |
| Aals-IDR-1+Vac | 43.8 | 15.24±1.42 | 7/10 | 103.9 |
| Aals-IDR-1018+Vac | 43.6 | 15.36±1.52 | 6/10 | 103.7 |
综上，AaLS-IDR-1047免疫增强肽配制成疫苗，免疫试验小鼠后，与各对照组相比，能够显著提高机体的抗体水平，并减少伪狂犬病毒在脑组织的载毒量，防止潜伏感染，提高保护率，对比其他Aals-IDR，只有AaLS-IDR-1047具有显著的免疫增强活性。
(3)AaLS-IDR-1047免疫增强试验
免疫前对每只小鼠称重，免疫后每周称重。每组第一次免疫28日龄SPF小鼠，肌肉免疫，0.2ml/只。每组21d后第二次免疫，肌肉免疫，0.2ml/只。通过倍比稀释抗原和不同毒株中和抗体检测验证AaLS-IDR-1047免疫增强能力。
实验分为七组：
PBS对照组：PBS作为水相与201佐剂乳化，使用时，0.2ml/只。
Vaccine组(伪狂犬病灭活疫苗免疫组)：该疫苗在使用时，疫苗中的伪狂犬病毒灭活抗原为2*107.0TCID50/ml。
AaLS-IDR-1047+Vac组：该疫苗在使用时，疫苗中的AaLS-IDR-1047为10μg/只，Vac(即伪狂犬灭活抗原)为2*107.0TCID50/ml。
AaLS-IDR-1047+两倍稀释Vac组：该疫苗在使用时，疫苗中的AaLS-IDR-1047为10μg/只，Vac(即伪狂犬灭活抗原)为1*107.0TCID50/ml。
AaLS-IDR-1047+三倍稀释Vac组：该疫苗在使用时，疫苗中的AaLS-IDR-1047为10μg/只，Vac(即伪狂犬灭活抗原)为0.66*107.0TCID50/ml。
AaLS-IDR-1047+四倍稀释Vac组：该疫苗在使用时，疫苗中的AaLS-IDR-1047为10μg/只，Vac(即伪狂犬灭活抗原)为0.5*107.0TCID50/ml。
AaLS-IDR-1047+五倍稀释Vac组：该疫苗在使用时，疫苗中的AaLS-IDR-1047为10μg/只，Vac(即伪狂犬灭活抗原)为0.4*107.0TCID50/ml。
二免后14d断尾采血分离血清，采用微量血清中和抗体实验(SMX、Ea、hSD毒株)检测伪狂犬抗体水平。二免采血后7d攻毒(伪狂犬SMX毒株，LD50＝19.5TCID50)20倍LD50200ul/只，取脑组织，组织切片免疫荧光染色(gB抗体)观察病毒粒子。
结果显示，二免后，添加AaLS-IDR-1047免疫增强肽的四倍稀释抗原组与伪狂犬病灭活疫苗对照组的血清中和抗体水平相当(图4)，证明添加了AaLS-IDR-1047免疫增强肽可以显著降低疫苗的抗原剂量至原疫苗的1/4，同时攻毒后小鼠脑组织伪狂犬gB抗体荧光结果(图5)显示，伪狂犬病灭活疫苗对照组脑组织仍有红色病毒粒子(图5中A)存在，添加AaLS-IDR-104免疫增强剂可以减少脑组织病毒粒子的潜伏(图5中B、C、D、E)。
综上，重组AaLS-IDR-1047免疫增强肽按合适比例添加进入倍比稀释抗原的伪狂犬病灭活疫苗水相并和油相201配制成疫苗，免疫试验小鼠后，与各对照组相比，能够显著提高机体的抗体水平，可减少配置疫苗时使用的抗原剂量，降低成本。并减少伪狂犬病毒在脑组织的病毒粒子，防止潜伏感染，提高保护率。
(4)猪验证AaLS-IDR-1047免疫增强肽试验
免疫前对每组猪编号标记。对35日龄的SPF猪进行第一次免疫，肌肉免疫，2ml/只；一免后21d第二次免疫，肌肉免疫，2ml/只。
实验分为四组：
PBS对照组：PBS作为水相与201佐剂乳化，使用时，2ml/只。
Vaccine组(伪狂犬灭活疫苗免疫组)：该疫苗在使用时，疫苗中的伪狂犬病毒灭活抗原为2*107.0TCID50/ml。
IDR-1047+Vac组：该疫苗在使用时，疫苗中的IDR-1047为10μg/只，Vac(即伪狂犬灭活抗原)为2*107.0TCID50/ml。
AaLS-IDR-1047+Vac组：该疫苗在使用时，疫苗中的AaLS-IDR-1047为10μg/只，Vac(即伪狂犬灭活抗原)为2*107.0TCID50/ml。
一免后21d前腔静脉采血分离血清，采用微量血清中和抗体实验(SMX毒株)检测伪狂犬抗体水平，同时采用双抗体一步夹心法酶联免疫吸附试验检测Th1型(干扰素-γ)和Th2型(IL-6)及肿瘤坏死因子(TNF-α)细胞因子的产生。二免后21天前腔静脉采血分离血清，采用微量血清中和抗体实验(SMX毒株)检测伪狂犬抗体水平，同时采用双抗体一步夹心法酶联免疫吸附试验检测Th1型(干扰素-γ)和Th2型(IL-6)及肿瘤坏死因子(TNF-α)细胞因子的产生。
结果显示，猪对于疫苗的吸收良好，体重增长正常(表2)，添加AaLS-IDR-1047免疫增强肽的疫苗组血清中和抗体水平最高(图6)，显著高于伪狂犬疫苗对照组和化学合成IDR组，二免21天后细胞因子检测结果显示(图7)，AaLS-IDR-1047显著调节Th1型(干扰素-γ)和Th2型(IL-6)及肿瘤坏死因子(TNF-α)细胞因子产生。干扰素-γ抑制病毒DNA和RNA的合成，进而抑制病毒复制，以及调节先天性和适应性免疫，发挥抗病毒作用。IL-6在获得性免疫应答中发挥重要作用，刺激抗体的产生和促使CD4+T细胞分化为效应T细胞。TNF-α提高中性粒细胞的吞噬能力，增加过氧化物阴离子产生，增强ADCC功能，刺激细胞脱颗粒和分泌髓过氧化物酶，刺激单核细胞和巨噬细胞分泌IL-1，并调节MHCⅡ类抗原的表达。
表2猪第二次免疫21天后伪狂犬灭活疫苗和不同浓度免疫增强剂组保护效果
| 分组 | 黏度/cP | 增重/kg | 吸收情况 |
| PBS | / | 16.72±2.22 | 吸收完全 |
| Vaccine | 32.0 | 17.11±1.24 | 吸收良好 |
| IDR-1047+Vac | 33.5 | 17.64±0.85 | 吸收良好 |
| AaLS-IDR-1047+Vac | 34.1 | 18.82±2.12 | 吸收良好 |
综上，重组AaLS-IDR-1047免疫增强肽按合适比例添加进入伪狂犬病疫苗水相并和油相201配制成疫苗，免疫试验猪后，与各对照组相比，能够显著提高机体的抗体水平，同时显著调节Th1型(干扰素-γ)和Th2型(IL-6)及肿瘤坏死因子(TNF-α)细胞因子产生。
Claims (9)
1. 一种人工合成的AaLS-IDR-1047免疫增强肽，所述免疫增强肽为SEQ ID NO.2所示。
2. 编码SEQ ID NO.2所示蛋白的基因。
3.含有权利要求2所述基因的表达载体。
4. 表达含有SEQ ID NO.2所示蛋白的重组微生物。
5. 权利要求1所述的AaLS-IDR-1047免疫增强肽的制备方法，包括：将编码SEQ IDNO.2所示蛋白的基因插入表达载体，转化大肠杆菌进行诱导表达。
6. 根据权利要求5所述的方法，所述的编码SEQ ID NO.2所示蛋白的基因为SEQ IDNO.1所示。
7.权利要求1所述的AaLS-IDR-1047免疫增强肽在制备疫苗免疫增强剂中的应用。
8.根据权利要求7所述的应用，所述的免疫增强剂的应用对象为小鼠和/或猪。
9.根据权利要求7所述的应用，所述的疫苗为猪伪狂犬灭活疫苗。
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| CA2290696A1 (en) * | 1997-05-19 | 1998-11-26 | Sumitomo Pharmaceuticals Company, Limited | Immunopotentiating composition |
| CN111956798A (zh) * | 2020-09-04 | 2020-11-20 | 江苏省农业科学院 | 用于猪伪狂犬灭活疫苗的复合免疫增强剂及其应用 |
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| Title |
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| 耿文学;唐波;华涛;侯继波;张道华;许家荣;: "免疫增强剂对猪伪狂犬灭活疫苗的免疫增强作用", 浙江农业学报, no. 11, 25 November 2016 (2016-11-25), pages 1828 - 1833 * |
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