CN110590910A - 能够增强细胞免疫应答和抗感染作用的合成肽 - Google Patents
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Abstract
本发明涉及了一种能够增强细胞免疫应答和抗感染作用的合成肽以及在诱导CD4+T细胞分化为Th1并产生高水平IFN‑γ中的应用。本发明还涉及了另一种能够增强细胞免疫应答和抗感染作用的合成肽以及在诱导CD4+T细胞分化为Th1和Th17并产生高水平IFN‑γ和IL‑17A中的应用。本发明筛选了两条能够增强细胞免疫应答和抗感染作用的合成肽，分别能够诱导CD4+T细胞分化为Th1并产生高水平IFN‑γ和诱导CD4+T细胞分化为Th1和Th17并产生高水平IFN‑γ和IL‑17A，可用于提高机体免疫力，提高抗感染能力。
Description
技术领域
本发明属于生物医学领域，涉及合成肽，特别涉及能够增强细胞免疫应答和抗感染作用的合成肽。
背景技术
抗感染过程中，细胞免疫应答发挥重要作用，抗原诱导后CD4+T细胞分化Th1、Th2和Th17等不同亚类，Th1细胞主要分泌IFN-γ，活化并增强巨噬细胞吞噬和杀灭胞内菌功能，促进B细胞转换抗体亚类；Th2细胞主要分泌IL-4、IL-5、IL-13等，促进体液免疫应答，刺激IgE、肥大细胞和嗜酸性粒细胞反应，清除蠕虫感染等；Th17细胞主要分泌IL-17、IL-22等，促进巨噬细胞、单核细胞和中性粒细胞迁移感染部位，发挥抗胞外菌和真菌感染作用。提高抗原诱导机体CD4+T细胞高水平表达特异性IFN-γ和IL-17A是疫苗研究的关键。
肽在机体体内所诱导的CD4+细胞免疫应答和产生IFN-γ和IL-17水平，能作免疫增强剂，增强表位疫苗的免疫原性，提高特异性免疫应答，对预防细菌感染发挥作用。因此，筛选能够增强细胞免疫应答和抗感染作用的合成肽显得尤为重要。
发明内容
本发明的第一目的是提供一种能够增强细胞免疫应答和抗感染作用的合成肽以及应用。
本发明的第二目的是提供另一种能够增强细胞免疫应答和抗感染作用的合成肽以及应用。
本发明通过以下技术方案来实现：
一、一种能够增强细胞免疫应答和抗感染作用的合成肽，其氨基酸序列如SEQ IDNo.1所示。
上述的合成肽在诱导CD4+T细胞分化为Th1并产生高水平IFN-γ中的应用。
二、一种能够增强细胞免疫应答和抗感染作用的合成肽，其氨基酸序列如SEQ IDNo.2所示。
上述的合成肽在诱导CD4+T细胞分化为Th1和Th17并产生高水平IFN-γ和IL-17A中的应用。
采用上述技术方案的积极效果：本发明筛选了两条能够增强细胞免疫应答和抗感染作用的合成肽，分别能够诱导CD4+T细胞分化为Th1并产生高水平IFN-γ和诱导CD4+T细胞分化为Th1和Th17并产生高水平IFN-γ和IL-17A，可用于提高机体免疫力，提高抗感染能力。
附图说明
图1是肽体内诱导CD4+T细胞增殖结果；
图2是肽体内诱导CD4+T细胞分泌IFN-γ水平；
图3是肽体内诱导CD4+T细胞分泌IL-17A水平。
具体实施方式
本发明中生物材料的来源：
所有的合成肽为上海楚肽生物科技有限公司合成。
下面结合具体的实施例对本发明的技术方案做进一步的说明，但不应理解成对本发明的限制：
实施例1
本实施例说明肽的合成。
利用在线软件模拟合成MHC-Ⅱ分子限制型肽，合成10条衍生肽序列详见表1。
表1肽的序列
实施例2
本实施例说明合成肽的效果。
1、免疫接种实验动物
取SPF级雌性6-8周龄BALB/c小鼠随机分组。饲养一周后，将合成肽分别与弗氏完全佐剂按体积比1:1比例乳化后，采用腿部肌肉注射方式，每只鼠注射100μg肽；初次免疫三周后，以等量的肽与弗氏不完全佐剂乳化后再次免疫接种小鼠。同时以无菌PBS与佐剂乳化作为对照。
2、制备抗原提呈细胞
无菌采取未经免疫接种的BALB/c小鼠脾脏，研磨制备细胞悬液，加入1mL红细胞裂解液于37℃处理2min，用10mL RPMI-1640培养液终止并清洗细胞三次，加入10mL RPMI-1640培养液(含10％胎牛血清、100U/mL青、链霉素)，重悬细胞，制成脾淋巴细胞悬液，置于CO2培养箱中(37℃、5％CO2)培养24h；弃去未贴壁细胞和细胞培养液，加入10mL含100μg丝裂霉素C的RPMI-1640培养液，置CO2培养箱中3h，将贴壁细胞吹下并清洗细胞3次以去除丝裂霉素C，使用1mL RPMI-1640培养液重悬细胞，稀释计数，并将细胞悬液稀释至1×106cells/mL备用。
3、CD4+T细胞的制备
将上述脾淋巴细胞悬液离心，弃掉上清液，依照CD4(L3T4)MicroBeads mouse产品说明进行操作，获得CD4+T细胞。使用1mL RPMI-1640培养液重悬细胞，稀释计数并将细胞悬液浓度调整至5×106cells/mL备用。
4、CD4+T细胞增殖试验
向96孔细胞培养板中加入各100μL调整好浓度的抗原提呈细胞和CD4+T细胞悬液及5μL浓度为1mg/mL的诱导物，使用等剂量PBS作为阴性对照，将96孔细胞培养板置于CO2培养箱中培养24h；再向每孔加入10μL的CCK-8溶液，将96孔细胞培养板置于CO2培养箱中孵育4h，使用酶标仪测定450nm波长处样品吸光度，并计算诱导指数(SI值)，确定CD4+T细胞增殖情况。SI＝(OD450实验组-OD450空白组)/(OD450未诱导-OD450空白组)
5、细胞因子检测
将已与诱导物及抗原提呈细胞共培养48h的CD4+T细胞悬液转移至2mL EP管中，2200r/min离心20min，收集上清，按照达科为产品Mouse IL-4、IFN-γ和IL-17A PrecoatedELISA Kit操作过程进行检测。
6、统计学分析
所有的数据进行生物统计学分析，采用SPSS软件进单因素方差分析，组间差异采用t检验，其中*表示p＜0.05，**表示p＜0.01，***p＜0.001，有统计学意义。
结果如下：
1、合成肽诱导CD4+T细胞增殖结果
肽免疫接种的BALB/c小鼠脾脏CD4+T细胞的特异性SI检测，结果各肽均可刺激CD4+T细胞不同程度增殖，其中衍生肽6、肽1、肽2和肽10刺激CD4+T细胞增殖水平较高，SI均大于1.5；其他衍生肽刺激CD4+T细胞，SI均小于1.5。结果详见图1。
2、肽体内诱导CD4+T细胞分泌细胞因子结果
肽免疫接种小鼠的脾脏分选得到CD4+T细胞，经各肽刺激后检测其诱导的特异性CD4+T细胞分泌的IFN-γ、IL-4和IL-17水平。结果，肽1、肽2诱导产生的IFN-γ水平明显升高，肽1产生IFN-γ水平显著高于肽2诱导的水平；结果详见图2。肽2诱导产生高水平的IL-17，与其它肽相比差异显著(p＜0.001)；结果详见图3。肽1能诱导更高水平的IFN-γ，但IL-17水平下降；肽2不仅能够诱导更高水平的IFN-γ，也能诱导更高水平的IL-17。
综上，本发明筛选了两条能够增强细胞免疫应答和抗感染作用的合成肽，分别能够诱导CD4+T细胞分化为Th1并产生高水平IFN-γ和诱导CD4+T细胞分化为Th1和Th17并产生高水平IFN-γ和IL-17A，可用于提高机体免疫力，提高抗感染能力。
序列表
<110> 黑龙江八一农垦大学
<120> 能够增强细胞免疫应答和抗感染作用的合成肽
<130> B012
<141> 2019-10-27
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Arg Asn Phe Gly Gly Phe Lys Ser Ala Arg Leu Leu Arg Pro Ala Lys
1 5 10 15
Gly Thr Thr Tyr
20
<210> 2
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Arg Asn Phe Gly Gly Phe Lys Ser Tyr Arg Leu Leu Ala Pro Ala Lys
1 5 10 15
Gly Thr Thr Tyr
20
<210> 3
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Arg Asn Phe Gly Ala Phe Lys Ser Tyr Arg Leu Leu Arg Pro Ala Lys
1 5 10 15
Gly Thr Thr Tyr
20
<210> 4
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Arg Asn Phe Gly Gly Ala Lys Ser Tyr Arg Leu Leu Arg Pro Ala Lys
1 5 10 15
Gly Thr Thr Tyr
20
<210> 5
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Arg Asn Phe Gly Gly Phe Ala Ser Tyr Arg Leu Leu Arg Pro Ala Lys
1 5 10 15
Gly Thr Thr Tyr
20
<210> 6
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Arg Asn Phe Gly Gly Phe Lys Ala Tyr Arg Leu Leu Arg Pro Ala Lys
1 5 10 15
Gly Thr Thr Tyr
20
<210> 7
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Arg Asn Phe Gly Gly Phe Lys Ser Tyr Ala Leu Leu Arg Pro Ala Lys
1 5 10 15
Gly Thr Thr Tyr
20
<210> 8
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Arg Asn Phe Gly Gly Phe Lys Ser Tyr Arg Leu Ala Arg Pro Ala Lys
1 5 10 15
Gly Thr Thr Tyr
20
<210> 9
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Arg Asn Phe Gly Gly Phe Lys Ser Tyr Arg Leu Leu Arg Ala Ala Lys
1 5 10 15
Gly Thr Thr Tyr
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<210> 10
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Arg Asn Phe Gly Gly Phe Lys Ser Tyr Arg Leu Leu Arg Pro Ala Ala
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Gly Thr Thr Tyr
20
Claims (4)
1.一种能够增强细胞免疫应答和抗感染作用的合成肽，其氨基酸序列如SEQ ID No.1所示。
2.由权利要求1所述的合成肽在诱导CD4+T细胞分化为Th1并产生高水平IFN-γ中的应用。
3.一种能够增强细胞免疫应答和抗感染作用的合成肽，其氨基酸序列如SEQ ID No.2所示。
4.权利要求2所述的合成肽在诱导CD4+T细胞分化为Th1和Th17并产生高水平IFN-γ和IL-17A中的应用。
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| WO2006032472A2 (en) * | 2004-09-22 | 2006-03-30 | Glaxosmithkline Biologicals S.A. | Immunogenic composition for use in vaccination against staphylococcei |
| CN105254719A (zh) * | 2015-10-21 | 2016-01-20 | 黑龙江八一农垦大学 | 金黄色葡萄球菌trap蛋白的cd4+t细胞表位鉴定及其重组表位疫苗 |
| CN109575141A (zh) * | 2017-09-29 | 2019-04-05 | 苏州工业园区唯可达生物科技有限公司 | 一种cd4辅助性t细胞表位融合肽及其疫苗 |
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| WO2006032472A2 (en) * | 2004-09-22 | 2006-03-30 | Glaxosmithkline Biologicals S.A. | Immunogenic composition for use in vaccination against staphylococcei |
| CN105254719A (zh) * | 2015-10-21 | 2016-01-20 | 黑龙江八一农垦大学 | 金黄色葡萄球菌trap蛋白的cd4+t细胞表位鉴定及其重组表位疫苗 |
| CN109575141A (zh) * | 2017-09-29 | 2019-04-05 | 苏州工业园区唯可达生物科技有限公司 | 一种cd4辅助性t细胞表位融合肽及其疫苗 |
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| CN114524880A (zh) * | 2022-02-10 | 2022-05-24 | 黑龙江八一农垦大学 | 一种能够增强免疫原性和免疫保护作用的rtg融合蛋白 |
| CN114524880B (zh) * | 2022-02-10 | 2023-09-19 | 黑龙江八一农垦大学 | 一种能够增强免疫原性和免疫保护作用的rtg融合蛋白 |
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