CN116396974B - 非洲猪瘟病毒抗原蛋白重组表达载体、重组植物乳酸菌及其制备方法和应用 - Google Patents
非洲猪瘟病毒抗原蛋白重组表达载体、重组植物乳酸菌及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及微生物技术领域,具体涉及一种非洲猪瘟病毒抗原蛋白重组表达载体、重组植物乳酸菌及其制备方法与应用,所述非洲猪瘟病毒抗原蛋白重组表达载体包含P54、Fc、P30和EGP基因片段;其中,P54与P30为非洲猪瘟病毒基因II型SY‑18毒株中编码参与对宿主吸附和内化作用相关的胞膜结构蛋白基因序列;所述Fc为猪源IgG3Fc基因序列,EGP为肠道细胞HSPGs受体靶向配体多肽序列。本发明获得的重组植物乳杆菌能高效表达P54/P30抗原蛋白,具有良好的免疫原性。同时与原始菌株L.Plantarum NC8相比,能诱导黏膜免疫、细胞免疫和体液免疫应答,具有良好的实际应用价值。
Description
本案为申请号202210157630.4案件的分案申请(申请日:2022年2月21日,发明创造名称:非洲猪瘟病毒抗原蛋白重组表达载体、重组植物乳酸菌及其制备方法和应用)。
技术领域
本发明涉及微生物技术领域,具体涉及一种非洲猪瘟病毒抗原蛋白重组表达载体、重组植物乳酸菌及其制备方法和应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)引发的急性、高度接触性、烈性传染病,死亡率高。由于目前仍未有有效商品化疫苗、特效药物对该疫病进行预防、治疗,非洲猪瘟病毒在宿主间传播过程中发生变异导致最近出现毒力变弱新毒株,病症轻且潜伏期长,一旦感染短期发现相对困难,非瘟疫情防控形势仍然复杂。
目前针对非洲猪瘟疫病预防的研究已有多种疫苗形式,但ASFV不同于其他核质大病毒之处在于其具有多层结构和整体二十面体形态,免疫生物学具有许多未知特征,有关感染性病毒粒子的组成和结构以及负责识别和诱导保护性免疫反应的病毒蛋白方面的知识差距很大,这就阻碍了有效疫苗的开发。研究发现ASFV p54、p30、CD2v、p12、p17和p72等结构蛋白参病毒与对宿主吸附和内化作用,并且P54蛋白位于ASFV病毒颗粒脂类外膜上,通过与动力蛋白轻链结合控制宿主细胞内的运输功能,在病毒感染过程中发挥重要作用,此外这些结构蛋白具有较好的免疫原性和保守性,能诱导中和抗体的产生和抑制病毒的内化作用,P54和P30蛋白已被作为ASFV疫苗的主要候选抗原。尽管有报道杆状病毒载体或痘苗病毒载体表面展示P54和P30抗原免疫可延迟ASF临床症状的发生,还能降低血液和淋巴组织中的病毒基因组载量,同时免疫后的仔猪可在E75毒株的攻击下存活,但是却不能抵抗高致病性基因型毒株的攻击。
分子流行病学研究表明传入中国的非洲猪瘟病毒属基因Ⅱ型,研究能够用于II型ASFV毒株预防的疫苗显得尤为重要。非洲猪瘟病毒可以编码上百种蛋白,其中E120R基因编码的结构蛋白p14.5被鉴定为该病毒的关键毒力因子和晚期表达蛋白,并能够引起机体产生体液免疫。传统的疫苗方法,如灭活病毒,已被证明是无效的。更多成功的是通过自然衰减分离物或修饰活病毒。然而,一般只针对同一基因型的同源菌株有保护作用,对异源病毒的感染无效。此外,减毒活株往往有相关的副作用,如皮肤病变和关节肿胀,无法作为安全的疫苗使用。活病毒疫苗也可能导致慢性或持续感染,并有毒力返强的风险。减毒活疫苗的另一个问题是缺乏稳定的生产细胞系,因为ASFV优先在原代单核或巨噬细胞中复制。
ASFV主要通过仔猪消化道和呼吸道感染其黏膜部位,其中消化道黏膜被认为是ASFV主要感染部位。发明人发现,尽管非洲猪瘟病毒以气溶胶或直接接触的形式进行传播并感染宿主黏膜,但ASFV侵入黏膜屏障具体机制并不清楚,并且通过黏膜系统递送抗原的免疫途径来保护仔猪阻断非洲猪瘟病毒感染是否有效值得深入研究。
发明内容
针对现有技术中存在的问题,本发明的目的是提供一种非洲猪瘟病毒抗原蛋白重组表达载体、重组植物乳酸菌及其制备方法,本发明一方面以食品级植物乳杆菌L.Plantarum NC8为出发菌株,以ASFV基因II型SY-18强毒株序列为基础,根据保护性抗原位点的研究,将相关的保护性抗原位点片段与猪源免疫球蛋白IgG Fc片段、硫酸乙酰肝素多糖配体多肽串联,并按照植物乳杆菌密码子偏好进行优化,从而成功构建获得NC8-pSIP-pgsA’-P54-Fc-P30-EGP(NC8-pSIP409-PPFPP)菌株。经试验研究证明,本发明获得的重组植物乳杆菌能高效表达P54/P30抗原蛋白,具有良好的免疫原性。同时与原始菌株L.Plantarum NC8相比,能诱导黏膜免疫、细胞免疫和体液免疫应答,具有良好的实际应用价值。
本发明的另一方面,选用非洲猪瘟病毒编码p14.5蛋白的E120R基因以及鼠源的IL-33和一种基于霍乱毒素(CT)的酶活性CTA1亚单位和金黄色葡萄球菌蛋白A的D域二聚体人工构成的佐剂CTA1-DD构建三株新功能型重组植物乳杆菌,NC8-pLP-S-p14.5,NC8-pLP-S-p14.5-IL-33-Mus和NC8-pLP-S-CTA1-p14.5-D-D,研究结果证实了本发明的重组植物乳杆菌通过口服方式饲喂小鼠后,小鼠在体液免疫、细胞免疫以及黏膜免疫方面的提升效果。
为了实现上述目的,本发明的技术方案如下所述:
在本发明的第一方面,提供一种非洲猪瘟病毒抗原蛋白重组表达载体,所述重组表达载体包含P54、Fc、P30和EGP基因片段;
其中,P54与P30为非洲猪瘟病毒基因II型SY-18毒株中编码参与对宿主吸附和内化作用相关的胞膜结构蛋白基因序列;所述Fc为猪源IgG3 Fc基因序列,EGP为肠道细胞HSPGs受体靶向配体多肽序列。
在一种或多种实施方式中,所述重组表达载体为P54-Fc-P30-EGP(PFPP);
具体地,上述P54、Fc、P30和EGP基因片段通过与pgsA’基因串联表达,P54-Fc-P30-EGP核苷酸序列如SEQ ID NO.1所示;
更进一步地,P54-Fc-P30-EGP编码蛋白其氨基酸序列如SEQ ID NO.2所示。
在本发明的第二方面,提供一种重组乳杆菌,所述重组乳杆菌转化有上述非洲猪瘟病毒抗原蛋白重组表达载体。
为提高上述外源基因编码蛋白在重组植物乳杆菌载体表面呈递外源抗原作用,本发明的重组乳杆菌利用pgsA’基因片段,通过枯草芽孢杆菌膜蛋白pgsA’锚定的表面展示抗原的pSIP409为表达载体。
因此,本发明的具体实施方案其中之一,所述重组乳杆菌包含pgsA’锚定的P54、Fc、P30和EGP基因片段。
在一种具体的实施方式中,所述重组乳杆菌为NC8-pSIP409-pgsA’-PFPP。
在本发明的第三方面,提供一种上述重组乳杆菌的构建方法,所述构建方法包括:
S1、将P54、Fc、P30和EGP基因片段插入锚定表达载体pSIP409-pgsA’中,构建重组表达载体P54-Fc-P30-EGP;
S2、将步骤S1的重组表达载体转入植物乳杆菌中即得。
在一种具体的实施方式中,步骤S1中,P54-Fc-P30-EGP的核苷酸序列如SEQ IDNO.1所示;P54-Fc-P30-EGP编码蛋白其氨基酸序列如SEQ ID NO.2所示;
在一种具体的实施方式中,步骤S2中,所述植物乳杆菌具体为植物乳杆菌NC8。
在本发明的第四方面,提供一种非洲猪瘟病毒抗原蛋白重组表达载体,所述非洲猪瘟病毒抗原蛋白重组表达载体为:pLP-S-p14.5,pLP-S-p14.5-IL-33和pLP-S-CTA1-p14.5-D-D;
其中,p14.5为非洲猪瘟病毒编码p14.5蛋白的E120R基因序列;IL-33为鼠源的IL-33基因序列;CTA1-DD为:基于霍乱毒素(CT)的酶活性CTA1亚单位和金黄色葡萄球菌蛋白A的D域二聚体人工构成的佐剂CTA1-DD基因序列;
优选地,pLP-S-p14.5的核苷酸序列如SEQ ID NO.3所示;
pLP-S-p14.5-IL-33的核苷酸序列如SEQ ID NO.4所示;
pLP-S-CTA1-p14.5-D-D的核苷酸序列如SEQ ID NO.5所示;
进一步优选地,pLP-S-p14.5编码蛋白其氨基酸序列如SEQ ID NO.6所示;
pLP-S-p14.5-IL-33编码蛋白其氨基酸序列如SEQ ID NO.7所示;
pLP-S-CTA1-p14.5-D-D编码蛋白其氨基酸序列如SEQ ID NO.8所示;
在一种具体的实施方式中,所述非洲猪瘟病毒抗原蛋白重组表达载体的构建方法为:
选取编码P14.5蛋白基因(E120R)序列,同时将两种佐剂序列IL-33和CTA1-DD优化后分别置于E120R基因5'3'端(CTA1-DD)与3'端(IL-33),P14.5,CTA1-P14.5-D-D,P14.5-IL-33-Mus序列合成后插入以erm为筛选标记单锚定表达载体pLP-S中,得到三个非洲猪瘟病毒抗原蛋白重组表达载体pLP-S-p14.5,pLP-S-p14.5-IL-33以及pLP-S-CTA1-p14.5-D-D。
蛋白p14.5在感染后期合成,位于病毒工厂中,是病毒粒子从病毒工厂转移到质膜时所必需的蛋白质,与蛋白P72相结合共同组成病毒的衣壳,并可以引起机体产生体液免疫。有研究认为蛋白p14.5可能在ASFV DNA的包埋过程中起作用。
疫苗佐剂是能够非特异性地改变或增强机体对抗原的特异性免疫应答、发挥辅助作用的一类物质。佐剂能够诱发机体产生长期、高效的特异性免疫反应,提高机体保护能力,同时又能减少免疫物质的用量,降低疫苗的生产成本;CTA1-DD是一种基于霍乱毒素的酶活性CTA1亚单位和金黄色葡萄球菌蛋白A的D域二聚体人工构成的佐剂,这种分子是无毒的,是一种安全的佐剂。它能够刺激强大而平衡的CD4+T细胞反应,极大地增强特异性抗体的产生;在全身免疫和黏膜免疫后,可以有效地成熟在外周淋巴结中的FDCs(滤泡树突状细胞),增加生发中心B细胞和Tfh的反应。
IL-33是一个多功能基因,在细胞发生损伤时分泌,在调节免疫中发挥作用,可以与DC直接反应,刺激幼稚T细胞向Th2或辅助性T细胞的分化;研究证明,在病毒感染期间,IL-33可以通过促进NK和NKT细胞的扩张以及增强Th1和CD8+T细胞应答,来帮助清除病毒。
S-层蛋白为细胞最外侧表面的单分子晶状结构蛋白,在许多古细菌中被认为是细胞膜的最外层结构,利用此蛋白表面展示外源蛋白;源于自乳酸杆菌的S-层蛋白不但具备在锚定表达外源蛋白的能力,还具有粘附特性,也可以作为刺激机体产生免疫应答的佐剂。
在本发明的第五方面,提供一种重组植物乳杆菌,所述重组植物乳杆菌为转化有第一方面所述非洲猪瘟病毒抗原蛋白重组表达载体,所述重组植物乳杆菌包括:NC8-pLP-S-p14.5,NC8-pLP-S-p14.5-IL-33-Mus和NC8-pLP-S-CTA1-p14.5-D-D。
在本发明的第六方面,提供一种第二方面所述重组植物乳杆菌的构建方法,所述构建方法包括:
S1、将p14.5基因、IL-33和佐剂CTA1-DD基因片段插入以erm为筛选标记单锚定表达载体pLP-S中,构建重组表达载体pLP-S-p14.5,pLP-S-p14.5-IL-33以及pLP-S-CTA1-p14.5-D-D;
S2、获得植物乳杆菌NC8感受态;
S3、将步骤S1的重组表达载体电转化至S2得到的物乳杆菌NC8感受态中即得。
在本发明的第七方面,提供一种菌剂,其包括上述重组植物乳杆菌或其发酵物或其代谢产物;
所述菌剂为液体或固体,优选为固体,进一步优选为冻干粉剂。
本发明所述的代谢产物包括菌体胞内代谢产物和/或胞外代谢产物。
术语“发酵物”用于指代发酵产品。相应的发酵物可以是从发酵培养重组植物乳杆菌的过程获得的液体,因此,也可称为发酵液;液体可以含有细菌(菌体),但并不必然需要含有细菌。液体优选的含有由本发明的重组植物乳杆菌细菌产生的代谢物。
以及,在本发明的实施方式中,包含菌体的发酵液或培养液经离心、过滤、沉降或所属领域中已知的其他手段将在发酵液或培养液中生长的菌体细胞与液体分离,去除菌体细胞时所剩余的液体为“上清液”(在本发明的实施方式中,上清液被标记为CFS),并且在本发明中,上清液内含有本发明重组植物乳杆菌的胞外代谢产物。在本发明的实施方式中,所述菌剂也可以包含该上清液。
以及,在本发明的实施方式中,包含菌体的发酵液或培养液经离心、过滤、沉降或所属领域中已知的其他手段将在发酵液或培养液中生长的菌体细胞与液体分离获取菌体,菌体可进行破碎获取菌体破碎物,破碎方式可以为超声(比如冰浴超声破碎细胞)或者本领域已知的其他手段,或者,更进一步的,对该菌体破碎物离心收集上清液,该上清液记为无细胞提取物,并且在本发明中,该菌体破碎物或无细胞提取物内含有本发明重组植物乳杆菌的胞内代谢产物。在本发明的实施方式中,所述菌剂也可以包含该菌体破碎物或无细胞提取物。
在本发明的第八方面,提供一种上述重组植物乳杆菌和/或所述菌剂在制备抗病毒产品中的应用。
所述重组植物乳杆菌和/或所述菌剂可以提高肠道对重组乳酸菌递送ASFV抗原蛋白的吸收作用,进而诱导高效黏膜免疫应答反应。
在本发明的第九方面,提供一种抗病毒产品,其特征在于,所述抗病毒产品包括上述重组植物乳杆菌和/所述菌剂;
优选的,所述产品为动物疫苗、饲料添加剂或饲料;
进一步优选的,所述动物疫苗为猪疫苗,所述疫苗剂型可以为口服冻干粉剂;
所述病毒包括非洲猪瘟病毒(ASFV)。
1.P54-Fc-P30-EGP核苷酸序(SEQ ID NO.1)
ATGGATAGTGAATTTTTTCAACCGGTTTATCCGCGTCATTATGGTGAATGTCTGAGTCCGGTTACGACGCCGAACTTTTTTAGTACGCACATGTATACCATTCTGATTGCGATTGTTGTTCTGGTTATTATCATTATCGTTCTGATTTATCTGTTTAGTAGTCGTAAAAAGAAAGCGGCGGCCATTGAAGAGGAAGATATTCAATTTATTAATCCGTATCAAGATCAACAATGGGTTGAAGTTACGCCGCAACCGGGTACGAGTAAACCGGCGGGTGCGACGACGGCGAGTGTTGGTAAGCCGGTTACGGGTCGTCCGGCCACGAATCGCCCGGCGACGAATAAACCGGTTACGGATAATCCGGTTACGGACCGTCTGGTGATGGCCACGGGTGGCCCGGCGGCCGCGCCGGCGGCCGCGAGTGCGCCGGCCCATCCGGCGGAACCGTATACGACGGTGACGACGCAGAATACGGCGAGTCAAACGATGAGTGCGATTGAAAATCTGCGTCAACGTAATACGTATACGCATAAAGATCTGGAAAATAGTCTGGGTAGTGGTGGTGGCGGTAGTGGTGGCGGTGGTAGCGGTAGTGACATTGAACCGCCGACGCCGATTTGTCCGGAAATTTGTAGTTGTCCGGCGGCGGAAGTTCTGGGTGCGCCGAGTGTTTTTCTGTTTCCGCCGAAACCGAAAGATATTCTGATGATTAGTCGTACGCCGAAAGTTACGTGTGTTGTGGTTGATGTTAGTCAAGAAGAAGCGGAAGTTCAATTTAGTTGGTATGTTGATGGTGTTCAACTGTATACGGCGCAAACGCGTCCGATGGAAGAACAATTTAATAGTACGTATCGTGTTGTTAGTGTTCTGCCGATTCAACATCAAGATTGGCTGAAAGGTAAAGCGTTTGCGTGTGCGGTTAATAATAAAGATCTGCTGAGTCCGATTACGCGTACGATTAGTAAAGCGACGGGTCCGAGTCGTGTTCCGCAAGTTTATACGCTGCCGCCGGCGTGGGAAGAACTGAGTAAAAGTAAAGTTAGTATTACGTGTCTGGTTACGGGTTTTTATCCGCCGGATATTGATGTTGAATGGCAAAGTAATGGTCAACAAGAACCGGAAGGTAATTATCGTACGACGCCGCCGCAACAAGATGTTGATGGTACGTATTTTCTGTATAGTAAACTGGCGGTTGATAAAGTTCGTTGGCAACGTGGTGATCTGTTTCAATGTGCGGTTATGCATGAAGCGCTGCATAATCACTATACGCAAAAAAGTATTAGTAAAACGCAAGGTAAAGAAGCGGCCGCGAAAGAAGCCGCGGCGAAAGAAGCGGCGGCCAAGGAAGCCGCGGCGAAGATGGATTTTATTCTGAATATTAGTATGAAAATGGAAGTTATTTTTAAAACGGATCTGCGTAGTAGCAGTCAAGTTGTTTTTCATGCGGGTAGTCTGTACAATTGGTTTAGTGTTGAAATTATTAATAGTGGTCGTATTGTTACGACGGCGATTAAAACGCTGCTGAGTACGGTTAAATATGATATTGTTAAAAGTGCGCGTATTTATGCGGGTCAAGGTTATACGGAACATCAAGCGCAAGAAGAATGGAATATGATTCTGCATGTTCTGTTTGAAGAGGAAACGGAAAGTAGTGCGAGTAGTGAAAATATTCATGAGAAAAATGATAATGAAACGAATGAATGTACGAGTAGTTTTGAAACGCTGTTTGAACAAGAACCGAGTAGTGAAGTTCCGAAAGATAGTAAACTGTATATGCTGGCGCAAAAAACGGTTCAACATATTGAACAATATGGTAAAGCGCCGGATTTTAATAAAGTTATTCGTGCGCATAATTTTATTCAAACGATTTATGGTACGCCGCTGAAAGAAGAGGAAAAAGAAGTTGTTCGTCTGATGGTTATTAAACTGCTGAAGAAAATTAACTTTTTTCTGACGTATATTGGTAGTGGTGGTGGCGGTAGTGGTAGTAAACGTAAAAAGAAAGGTAAAGGTCTGGGTAAAAAACGTGATCCGTGTCTGCGTAAATATAAA
2.P54-Fc-P30-EGP氨基酸序列(SEQ ID NO.2)
MDSEFFQPVYPRHYGECLSPVTTPNFFSTHMYTILIAIVVLVIIIIVLIYLFSSRKKKAAAIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSLGSGGGGSGGGGSGSDIEPPTPICPEICSCPAAEVLGAPSVFLFPPKPKDILMISRTPKVTCVVVDVSQEEAEVQFSWYVDGVQLYTAQTRPMEEQFNSTYRVVSVLPIQHQDWLKGKAFACAVNNKDLLSPITRTISKATGPSRVPQVYTLPPAWEELSKSKVSITCLVTGFYPPDIDVEWQSNGQQEPEGNYRTTPPQQDVDGTYFLYSKLAVDKVRWQRGDLFQCAVMHEALHNHYTQKSISKTQGKEAAAKEAAAKEAAAKEAAAKMDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKINFFLTYIGSGGGGSGSKRKKKGKGLGKKRDPCLRKYK
3 p14.5核苷酸序列(SEQ ID NO.3)
ATGGCTGATTTTAATTCACCCATACAATATCTCAAAGAAGACTCTCGCGATCGTACCTCGATCGGCAGCTTGGAATATGATGAAAATGCAGATACGATGATTCCGAGCTTTGCGGCGGGTCTGGAAGAGTTCGAGCCGATTCCAGACTACGACCCGACCACGTCCACCTCCCTTTACAGCCAGCTGACCCATAACATGGAAAAGATCGCCGAGGAGGAGGACTCTAACTTCCTGCATGATACCCGTGAATTCACTTCATTGGTTCCGGACGAGGCGGACAACAAGCCGGAAGATGATGAGGAAAGCGGTGCTAAACCGAAAAAGAAGAAGCACCTGTTTCCGAAGCTGAGCAGCCATAAAAGCAAA
4 p14.5-IL-33-Mus核苷酸序列(SEQIDNO.4)
ATGGCTGATTTTAATTCACCCATACAATATTTGAAGGAGGACAGCAGAGATCGTACCTCCATCGGTAGCCTGGAGTATGATGAGAACGCCGACACGATGATTCCGAGCTTTGCGGCTGGTCTGGAAGAGTTCGAGCCGATTCCGGATTACGACCCAACGACCTCCACCTCCCTGTATAGCCAACTCACCCATAATATGGAAAAGATCGCCGAAGAGGAGGACTCTAACTTCCTGCACGATACGCGTGAATTCACCTCTTTAGTTCCGGATGAGGCGGATAATAAGCCGGAAGACGACGAAGAAAGCGGTGCTAAACCGAAGAAGAAGAAGCACCTGTTTCCGAAACTGAGCTCCCATAAAAGCAAAGGTTCTGGCGGTGGCGGCTCTGGCGGCGGCGGGTCAGGTTCCATGCGTCCGCGTATGAAATACTCCAATAGCAAGATTAGCCCGGCGAAATTCAGCAGCACTGCCGGTGAACGTAGCGTCCCACCGTGCAAAATCCGCCGTAGTCAGCAGAAAACCAAAGAGTTCTGCCATGTTTACTGCATGCGCCTGCGCTCGGGCCTGACCATTCGTAAAGAAACCTCTTACTTCCGCAAAGAGCCGACTAAGCGCTACTCCCTTAAAAGCGGTACCAAACATGAAGAGAACTTTAGCGCATATCCGCGTGATAGCCGTAAGCGCAGCTTGCTGGGTTCTATCCAAGCGTTTGCGGCGAGTGTGGATACCCTGTCGATCCAGGGTACATCGTTGCTGACCCAGAGCCCGGCGAGCTTATCTACCTATAACGACCAGAGCGTCAGCTTCGTTTTGGAGAACGGTTGTTATGTGATCAACGTGGACGATTCCGGCAAGGACCAAGAACAAGATCAAGTGTTGCTGCGTTACTATGAAAGCCCGTGCCCGGCTTCGCAAAGCGGTGATGGTGTGGACGGCAAAAAGCTGATGGTTAATATGAGCAGCATCAAGGACACCGACATCTGGCTGCATGCAAACGATAAAGACTACTCTGTGGAACTGCAGCGTGGTGATGTTAGCCCGCCTGAACAGGCGTTTTTTGTACTTCACAAAAAGAGCTCAGATTTTGTGTCCTTCGAGTGCAAGAACTTGCCGGGTACGTACATTGGTGTTAAAGACAACCAGCTGGCTCTGGTTGAAGAGAAGGACGAGAGCTGTAATAATATTATGTTCAAACTGTCGAAAATT
5 CTA1-p14.5-D-D核苷酸序列(SEQ ID NO.5)
ATGAATGATGACAAACTATATAGGGCTGATAGCCGTCCGCCAGATGAGATCAAACAATCTGGTGGTCTGATGCCGCGCGGTCAGTCCGAATACTTCGATAGAGGAACCCAGATGAACATTAACCTGTACGACCACGCGACCCAGACCGGTTTTGTGCGTCATGATGATGGCTACGTCAGCACTTCTATTAGCTTACGTAGCGCGCATTTGGTTGGTCAGGAGGTTAGCGCGCTGGGTGGTATCCCGTATAGCCAGATCTATGGTTCGTACCGCGTGCATTTTGGTGTTCTGGACGAACAGCTGCATCGTAACCGTGGTTATCGTTATTACTCTAACCTCGACATCCCGCCGGCGGCCGACGGCTACGGCCTAGCGGGTTTCCCGCCGGAGCACCGCGCATGGCGCCAGGAGCCGTGGATTCACCATGCTCCGCCAGGTTGCGGTAATGCCCCACGTAGCAGCGGCTCTGGCGGCGGTGGCTCAGGCGGTGGCGGTAGCGGTTCTATGGCTGACTTCAACAGCCCGATCCAGTATCTGAAAGAAGACTCTCGCGATCGTACCTCGATCGGTTCATTGGAATATGATGAGAATGCCGATACTATGATCCCGAGCTTTGCAGCGGGCCTGGAAGAATTTGAACCGATTCCGGATTATGATCCGACCACCTCGACGTCACTTTACAGTCAACTGACCCATAATATGGAAAAAATCGCTGAAGAGGAGGATTCCAACTTTCTGCACGATACGCGTGAGTTCACCAGCCTTGTTCCGGACGAGGCTGACAACAAACCGGAGGACGACGAGGAAAGCGGCGCAAAACCGAAAAAGAAGAAGCATCTGTTTCCGAAACTGAGCAGCCACAAAAGCAAGGGTTCCGGTGGCGGTGGTAGCGGCGGCGGTGGCTCTGGGAGCGCCGACGCACAGCAGAATAATTTCAACAAGGACCAACAAAGCGCGTTTTACGAAATTTTGAATATGCCGAATTTGAATGAGGCGCAACGCAACGGCTTCATCCAGTCCTTGAAAGACGATCCGTCCCAGAGCACCAACGTGTTGGGTGAGGCGAAAAAGCTGAATGAGTCCCAAGCGCCCAAGGGCAGCGGTGGCGGCGGTTCCGGTGGAGGCGGCAGTGGGAGCGCTGATGCGCAGCAAAACAACTTCAACAAAGACCAACAGTCTGCGTTCTACGAAATTCTGAACATGCCGAACCTGAACGAAGCACAGCGTAATGGTTTCATTCAAAGCCTGAAGGACGACCCGAGCCAATCTACGAACGTGCTGGGCGAAGCCAAAAAGCTGAACGAGAGCCAAGCGCCTAAG
6 p14.5氨基酸序列(SEQ ID NO.6)
MADFNSPIQYLKEDSRDRTSIGSLEYDENADTMIPSFAAGLEEFEPIPDYDPTTSTSLYSQLTHNMEKIAEEEDSNFLHDTREFTSLVPDEADNKPEDDEESGAKPKKKKHLFPKLSSHKSK
7 P14.5-IL-33-Mus氨基酸序列(SEQ ID NO.7)
MADFNSPIQYLKEDSRDRTSIGSLEYDENADTMIPSFAAGLEEFEPIPDYDPTTSTSLYSQLTHNMEKIAEEEDSNFLHDTREFTSLVPDEADNKPEDDEESGAKPKKKKHLFPKLSSHKSKGSGGGGSGGGGSGSMRPRMKYSNSKISPAKFSSTAGERSVPPCKIRRSQQKTKEFCHVYCMRLRSGLTIRKETSYFRKEPTKRYSLKSGTKHEENFSAYPRDSRKRSLLGSIQAFAASVDTLSIQGTSLLTQSPASLSTYNDQSVSFVLENGCYVINVDDSGKDQEQDQVLLRYYESPCPASQSGDGVDGKKLMVNMSSIKDTDIWLHANDKDYSVELQRGDVSPPEQAFFVLHKKSSDFVSFECKNLPGTYIGVKDNQLALVEEKDESCNNIMFKLSKI
8 CTA1-P14.5-D-D氨基酸序列(SEQ ID NO.8)
MNDDKLYRADSRPPDEIKQSGGLMPRGQSEYFDRGTQMNINLYDHATQTGFVRHDDGYVSTSISLRSAHLVGQEVSALGGIPYSQIYGSYRVHFGVLDEQLHRNRGYRYYSNLDIPPAADGYGLAGFPPEHRAWRQEPWIHHAPPGCGNAPRSSGSGGGGSGGGGSGSMADFNSPIQYLKEDSRDRTSIGSLEYDENADTMIPSFAAGLEEFEPIPDYDPTTSTSLYSQLTHNMEKIAEEEDSNFLHDTREFTSLVPDEADNKPEDDEESGAKPKKKKHLFPKLSSHKSKGSGGGGSGGGGSGSADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKGSGGGGSGGGGSGSADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK
本发明的具体实施方式具有以下有益效果:
一方面,构建两株重组植物乳酸菌NC8-pSIP409-pgsA’-PFPP与NC8-pSIP409-pgsA’-PmFPP,对小鼠进行了口服灌胃免疫实验,对比PBS以及NC8组,免疫一周后,检测重组植物乳酸菌免疫后对小鼠细胞免疫的影响,结果显示两种重组植物乳酸菌均可以诱导小鼠脾脏产生CD4+IL-4+和CD8+IFN-γ+细胞,表明重组植物乳酸菌表达的融合蛋白作为诱导机体细胞免疫的抗原,成功诱导了T淋巴细胞的反应,可能两个T细胞亚群均起到保护作用;
使用流式细胞术检测了重组植物乳酸菌免疫后小鼠派尔集合淋巴结B细胞中IgA的含量,结果显示重组植物乳酸菌中B220+IgA+的数量显著高于PBS和NC8组,表明在重组植物乳酸菌的刺激下抗体分泌量显著增加,成功活化了B淋巴细胞的反应;
两种重组植物乳酸菌在口服灌胃后均能够刺激派尔集合淋巴结中CD80+和CD86+表面标志的活化,表明两种重组植物乳酸菌均能够使树突状细胞活化,使其进一步发挥其抗原摄取呈递功能;
通过灌胃的方式免疫小鼠,两株重组植物乳酸菌能更好的刺激DC细胞的活化,诱导小鼠产生细胞因子,促进T细胞增殖,提高B细胞的活化水平,为生发中心的形成提供了条件,并促进了IgA的表达。
另一方面,本发明成功构建了NC8-pLP-S-p14.5,NC8-pLP-S-p14.5-IL-33-Mus和NC8-pLP-S-CTA1-p14.5-D-D三种重组植物乳杆菌,并验证了它们在体外的成功表达;由于乳酸菌可以在小鼠肠道内定殖,并能在肠道环境下可以刺激乳酸菌表达p54蛋白以及p54-IL-33、CTA1-p14.5-D-D融合蛋白,机体T细胞识别p54蛋白以及p54-IL-21融合蛋白并产生特异性的抗体;用小鼠进行初步动物试验,结果可见饲喂重组植物乳杆菌对于小鼠的免疫效果具有一定的提升作用;
由流式细胞术实验可见饲喂重组植物乳杆菌组小鼠的PP结淋巴细胞中B220和IgA的分泌的量以及11C+CD80和11C+CD86的含量大于对照组;脾脏中CD4+IFN-γ+CD4+IL-4和的CD8+IFN-γ+CD8+IL-4表达量也有显著的增强。
本发明的实验结果都表明了当饲喂本发明的重组植物乳杆菌后,小鼠的免疫力有显著的提高。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为:实施例1中的pLP-S-p14.5质粒双酶切验证(M:DL15000 marker;1-4:第1-4号克隆酶切结果);
图2为:实施例1中的pLP-S-CTA1-p14.5-D-D质粒双酶切验证(M:DL15000 marker;1-2:第1-2号克隆酶切结果);
图3为:实施例1中的pLP-S-p14.5-IL-33-Mus质粒双酶切验证(M:DL10000marker;1:第1号克隆酶切结果);
图4为:实施例1中的成功构建好的NC8-pLP-S-p14.5质粒图谱;
图5为:实施例1中的成功构建好的NC8-pLP-S-p14.5-IL-33-Mus质粒图谱;
图6为:实施例1中的成功构建好的NC8-pLP-S-CTA1-p14.5-D-D质粒图谱;
图7为:实施例1中的p14.5 PCR产物电泳图(M:DL2000 marker;1:p14.5 pcr产物);
图8为:实施例1中的BL21-pET-28a-p14.5质粒双酶切验证(M:DL10000 marker;1:第1号克隆酶切结果);
图9为实施例1中的NC8-pLP-S、NC8-pLP-S-P14.5、NC8-pLP-S-P14.5-IL-33-Mus、NC8-pLP-S-CTA1-P14.5-D-D菌株生长曲线图;
图10为:实施例2中PBS组、NC8-pLP-S组、NC8-pLP-S-P14.5组、NC8-pLP-S-CTA1-P14.5-D-D组、NC8-pLP-S-P14.5-IL-33-Mus组脾脏淋巴细胞中分泌量图及柱状图;其中,(a)为PBS组的IFN-γ分泌量;(b)为NC8-pLP-S组的IFN-γ分泌量,(c)为NC8-pLP-S-P14.5组的IFN-γ分泌量,(d)为NC8-pLP-S-CTA1-P14.5-D-D组的IFN-γ分泌量,(e)为NC8-pLP-S-P14.5-IL-33-Mus组的IFN-γ分泌量;
图11为:实施例2中PBS组、NC8-pLP-S组、NC8-pLP-S-P14.5组、NC8-pLP-S-CTA1-P14.5-D-D组、NC8-pLP-S-P14.5-IL-33-Mus组的脾脏淋巴细胞中CD3+CD4+T细胞中IL-4分泌量图及柱状图,其中,(a)为PBS组的IL-4分泌量;(b)为NC8-pLP-S组的IL-4分泌量,(c)为NC8-pLP-S-P14.5组的IL-4分泌量,(d)为NC8-pLP-S-CTA1-P14.5-D-D组的IL-4分泌量,(e)为NC8-pLP-S-P14.5-IL-33-Mus组的IL-4分泌量;
图12为:实施例2中脾脏淋巴细胞中CD3+CD8+T细胞中IL-4分泌量;
图13为:实施例2中脾脏淋巴细胞中CD3+CD8+T细胞中IFN-γ分泌;
图14为:实施例2中脾派依氏集合淋巴结(PP)细胞中11C+80+含量;
图15为:实施例2中脾派依氏集合淋巴结(PP)细胞中11C+86+含量;
图16为:实施例2中脾派依氏集合淋巴结(PP)细胞中B220+IGA+含量。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本申请使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
下面结合具体的实施例对本发明作进一步的解释和说明。
实施例1
重组植物乳杆菌的构建及蛋白表达验证与纯化
1.1材料
1.1.1质粒
以非洲猪瘟病毒毒株BA71V为参考序列,选取毒株中编码P14.5蛋白基因(E120R)序列,由南京金斯瑞生物科技有限公司按照植物乳杆菌表达密码子优化合成。同时将两种佐剂序列优化后分别置于E120R基因5'3'端(CTA1-DD)与3'端(IL-33),P14.5,CTA1-P14.5-D-D,P14.5-IL-33-Mus序列合成后插入以erm为筛选标记单锚定表达载体pLP-S中,得到三个质粒pLP-S-p14.5,pLP-S-p14.5-IL-33以及pLP-S-CTA1-p14.5-D-D。
1.1.2菌株及质粒载体
NC8乳酸菌由实验室保存,乳酸菌表面展示系统(pLP-S)由实验室保存。
1.1.3主要试剂
限制性内切酶(SalI和XhoI)、SppIP购于宝生物工程大连有限公司;10×Hbuffer,DNAMarker(DL-15000、DL-10000)购于Takara生物公司;红霉素购于北京索莱宝科技有限公司;质粒小提试剂盒盒购于北京全式金生物公司;电击杯购自Bio-Rad公司;DNA凝胶回收试剂盒购于美国OmegaBio-Tek公司;诱导剂SppIP由实验室保存;BCA蛋白浓度测定试剂盒购于碧云天生物技术公司。
1.1.4主要试剂的配制
1)LB培养基配方:蛋白胨10g,酵母5g,NaCl 10g;
先溶于800mL蒸馏水,完全溶解后补充至1L,分装至试管或锥形瓶中,121℃高压灭菌20min备用(液体)。在配好的LB液体培养基中加入1.5%的琼脂粉,121℃高压灭菌20min备用(固体)。
2)MRS培养基配方:蛋白胨10g,酵母5g,葡萄糖10g,牛肉蛋白粉10g,醋酸钠5g,柠檬酸二铵2g,磷酸氢二钾2g,吐温-80 1mL,七水硫酸镁0.2g,一水硫酸锰0.05g;
先溶于800mL蒸馏水,完全溶解后补充至1L,用高压锅115℃灭菌15min,调节pH6.2~6.4(液体)。在配好的MRS液体培养基中加入1.5%的琼脂粉,121℃高压灭菌20min备用(固体)。
3)50×TAE电泳缓冲液(PH8.5)配方:Tris 242g,Na2EDTA·2H2O 37.2g;注意:溶于1000mL蒸馏水中,室温保存即可。
4)5×SDS电泳缓冲液配方:Tris 15.1g,Glycine 94g,SDS 5g;注意:溶于1000ml蒸馏水中,4℃保存。
5)TBST配方:NaCl 8.8g,1M/LTris-HCL(PH8.0)20mL,去离子水800mL,Tween-200.5mL;注意:溶于1000mL蒸馏水中,4℃保存。
6)转膜液配方:Glycine 2.9g,Tris 5.8g,SDS 0.37g,甲醇200mL,去离子水600mL;
注意:溶于1000ml蒸馏水中,4℃保存。
7)PBS缓冲液:PBS缓冲剂0.02M,蒸馏水1800mL;注意:121℃高压灭菌后室温保存。
8)电转化溶液
电击缓冲液:MgCl20.029 g、蔗糖34.21g、dH2O 80mL,以稀盐酸将溶液pH调至7.4,加入蒸馏水定容至100mL,121℃灭菌20min,4℃储存备用。
清洗缓冲液:Na3PO40.19 g、MgCl20.009g;dH2O 80mL以稀盐酸将pH调至7.4,加入蒸馏水定容至100mL,121℃灭菌20min,4℃储存备用。
9)蛋白电泳溶液(5×RunningBuffer):Tris粉末7.575g,甘氨酸粉末35.1g,SDS粉末2.5g,加ddH2O溶解均匀,最后定容至500mL,以1:5(v/v)使用。
10)30%丙烯酰胺溶液:丙烯酰胺87.6g,甲叉双丙烯酰胺2.4g,加ddH2O溶解混匀定容至300mL,褐色瓶盛装并避光保存。
11)1.5MTris-Cl,pH 8.8:Tris粉末56.013g加入ddH2O中混匀,缓慢滴加1MHCl调节溶液PH至8.8,最终ddH2O定容至300mL。
12)0.5M Tris-Cl,pH 6.8:Tris粉末36.342g加入dd H2O中混匀,缓慢滴加1M HCl调节溶液PH至6.8,最终ddH2O定容至300mL。
13)10%APS溶液:APS(过硫酸铵)粉末0.3g,加入dd H2O溶解混匀定容至5mL,分装(500μL/份)并于-20℃中冻存备用。
14)碱性磷酸酶(AP)缓冲液:NaCl 0.58g,MgCl2·6H2O 0.11g,Tris 1.21g,加入ddH2O搅拌混匀并定容至100mL,4℃存放备用。
1.2方法
1.2.1目的基因p14.5、IL-33-Mus、CTA1-DD的序列获取
通过NCBI获得p14.5与IL-33-Mus的核苷酸序列及氨基酸序列,通过文献查询获得CTA1-DD的核苷酸序列及氨基酸序列。
1.2.2质粒的构建
南京金斯瑞生物科技有限公司按照植物乳杆菌表达密码子优化P14.5,CTA1-P14.5-D-D,P14.5-IL-33-Mus序列,合成后插入以erm为筛选标记单锚定表达载体pLP-S中,得到三个质粒pLP-S-p14.5,pLP-S-p14.5-IL-33以及pLP-S-CTA1-p14.5-D-D。
1.2.3质粒双酶切及测序鉴定
(1)质粒样品送至吉林省库美生物技术公司进行测序鉴定。
(2)15μL酶切鉴定体系如下:质粒10.5μL,酶SalI 1.5μL,酶XhoI 1.5μL,Buffer1.5μL。
1.2.4酶切产物凝胶电泳试验
将酶切产物取1-10μL混上10×的Loading Buffer 2μL加入琼脂糖凝胶孔中,在120 V/20min的条件下进行电泳试验,电泳结束后取下凝胶放入照胶台,经台式紫外成像分析仪器可见大小与预期是否相符。
1.2.5植物乳杆菌NC8感受态的获得
(1)取-80℃冻存的植物乳杆菌NC8接种于5mL含Erm(5μg/mL)的MRS液体培养液中,置于厌氧工作站中培养;(2)含Erm(5μg/mL)的MRS固体培养基上均匀涂抹0.1mL培养后的菌液,置于厌氧工作站中培养至长出单菌落(16-18h);(3)取5mL Erm(5μg/mL)的MRS液体培养基(含2%Gly)接种固体平板上的单菌落,置于厌氧工作站中培养直至菌液OD600值为0.6-0.8;(4)取50mL含的MRS液体培养基(含2%Gly,5μg/mL Erm)接种培养1mL上述菌液,厌氧条件下继续培养直至菌液OD600值为0.3;(5)将上述收集的菌液冰浴10min,4℃条件下8000r/min离心5min,收集菌体沉淀;(6)将菌体沉淀以冰冷的2mL清洗缓冲液重悬,4℃条件下8000r/min离心10min,重复清洗两次,收集菌体沉淀;(7)将菌体沉淀以冰冷的200μL电击缓冲液重悬,冰浴10min后以每管100μL进行分装,液氮速冻后置于-80℃冰箱备用。
1.2.6将质粒电转化至植物乳杆菌NC8中
(1)分别取5μL重组质粒pLP-S-P14.5、pLP-S-P14.5-IL-33-Mus、pLP-S-CTA1-P14.5-D-D加入三管冰浴的100μL乳酸菌NC8感受态中,轻缓混匀,移入电击杯内,静置冰浴5min后,放入电转化仪(2.5Kv,6ms)中进行电击;
(2)完成电击后,继续冰浴3-5min,取800μLMRS培养液加入电击杯中,混匀后将电击杯内液体全部吸出至1.5mL的离心管中,封口膜封口后,置于厌氧工作站培养2h;
(3)取出ep管,4000rpm,离心3min,弃去上清液,留100μL用无菌的涂布棒均匀涂布于含Erm(5μg/mL)的MRS固体培养基上,厌氧培养直至长出大小合适的单菌落,约16-20h。
1.2.7重组植物乳杆菌质粒的提取及鉴定
将固体培养基中乳酸菌单菌落接种于含Erm(5μg/mL)的5mLMRS培养液中,厌氧条件下培养16-20h,用质粒小量提取试剂盒(使用溶菌酶破壁)进行质粒提取。提取的质粒使用SalI/XhoI进行双酶切,凝胶电泳鉴定质粒是否成功转入乳酸菌NC8中。
1.2.8重组植物乳杆菌菌生长曲线测定
a.将NC8-pLP-S-p14.5,NC8-pLP-S-p14.5-IL-33-Mus和
NC8-pLP-S-CTA1-p14.5-D-D分别划线于加入EM抗性的平板上,于37℃厌氧箱中培养16h后,选取大小适中的单菌落挑菌到5mL加入EM抗性的MRS试管中过夜活化培养;b.取活化的NC8菌液1:30接菌于50mL加入EM抗性的离心管中;待三组菌液OD值都为0.3-0.4之间时,加入乳酸菌诱导剂SppIP(12.5μL/5mL);c.每间隔1h从各组菌液中分别取2mL测OD600值,进行12小时的监测并绘制生长曲线。
1.2.9菌落滴板计数
经多次检测,当培养12h时NC8-pLP-S、NC8-pLP-S-p14.5、NC8-pLP-S-p14.5-IL-33-Mus和NC8-pLP-S-CTA1-p14.5-D-D菌液的OD值分别为2.09、1.64、1.42和1.59。分别梯度稀释106,107,108后取100μL进行滴板计数,总结发现0.1mL的NC8-pLP-S-p14.5有5.5×109个菌落数;0.5mL的NC8-pLP-S-p14.5有1×109个菌落数;0.8mL的NC8-pLP-S-p14.5-IL-33-Mus有1×109个菌落数;1.1mL的NC8-pLP-S-CTA1-p14.5-D-D有1×109个菌落数(小鼠饲喂乳杆菌最佳数量为0.5×109-1×109)
1.2.10重组植物乳杆菌的Westenblot检测
(1)蛋白样品处理
a.将保藏的菌液NC8-pLP-S-p14.5,NC8-pLP-S-p14.5-IL-33-Mus和NC8-pLP-S-CTA1-p14.5-D-D从-80℃冰箱中取出,接种于5mL含Erm(5μg/mL)的MRS液体,过夜培养;b.次日转接至50mL加入Erm(5μg/mL)的MRS液体中,37℃厌氧工作站培养1h后分别加入SppIP125μL(50mg/mL),诱导培养6h后,离心取沉淀用PBS洗3遍后分别用5mLPBS重悬;c.将样品在60W的条件下超声破碎处理20min,将细菌上清液和沉淀分别收集于Ep管中。
(2)Western blot
a.首先配置凝胶;b.NC8-pLP-S-p14.5,NC8-pLP-S-p14.5-IL-33-Mus和NC8-pLP-S-CTA1-p14.5-D-D分别取10μL菌液加5μL的5×上样缓冲液于100℃沸水中煮10min;c.将样品加入胶槽内,上层胶用80V跑45min,下层胶用150V跑30min;d.转膜预先将转膜滤纸在转膜液中浸润,取出凝胶,根据所需大小剪下一块大小合适的NC膜,注意边缘整齐,甲醇中浸泡30s激活后,再浸泡于转印缓冲液中;e.在转膜液中按照从上到下依次为海绵、滤纸、NC膜、凝胶、滤纸、海绵的顺序排放好于转印夹中,注意白色一侧在上,黑色在下,用小滚轮除去每层之间产生的气泡,NC膜一侧与正极相连,凝胶一侧与负极相连,恒流300mA/1h,转膜过程全程在冰上进行;f.封闭:取出NC膜,加入1×PBS迅速清洗一遍,目的是洗掉多余的电转液。再加入PBST溶液,室温封闭1h;g.抗体孵育:弃掉封闭液,1×PBST清洗两遍,1×PBS清洗一遍,5min/遍,洗掉多余的封闭液。加入适量的抗体稀释液中,置于摇床上4℃孵育过夜;h.ECT显色:将ECT显色试剂盒的A液与B液按照1:1比例混合均匀后,均匀涂抹于NC膜上放入AI 600仪器中进行显色,并获取图像结果。
1.2.11构建重组大肠杆菌BL21-Pet-28a-p14.5
(1)引物设计
为得到带有酶切位点Nco I和Xho I的p14.5基因片段设计上下游引物进行PCR扩增,在ASFV p514.5序列上游5'端添加Nco I酶切位点。
F:5'CATGCCATGGGTCGACATGGCTGATTTTAATTCACCC 3'
R:5'CTCGAGGTGGTGATGGTGGTGGTG 3'
(2)PCR扩增
PCR扩增体系如下:质粒(p14.5-pLP-S)1μL,PrimeSTAR Max 25μL,P14.5 F 2μL,P14.5 R 2μL,ddH2O 20μL,总共50μL;
PCR反应程序见表3
表3
步骤2-4重复35个循环;
(3)胶回收
扩增片段大小为402bp,胶回收。
(4)连接Peasy-Blunt-zero,转化至T1感受态中
a.将胶回收产物与载体4:1混合,即4μL PCR产物,1μL载体,在16℃条件下使用T4连接酶进行连接;b.将连接产物小心的加到T1感受态(冰上溶解)中,轻弹混匀后放置冰上30min;c.42℃水浴锅热激90S后置于冰上静置5min;d.向T1感受态中加入300μL平衡至室温的液体LB,37℃摇床培养1h;e.取出培养后的菌液,4000rpm离心5min,弃上清留100μL悬浮菌体沉淀后均匀涂抹在加入Kana抗性的LB平板上,将平板倒置于37℃温箱培养;
(5)挑菌鉴定质粒,酶切回收目的片段
a.从平板上挑取单菌落加入Kana抗性的液体LB试管中过夜培养,提取质粒进行双酶切鉴定和测序鉴定;
b.将双酶切后回收的目的片段与-20℃冰箱中保存;
(6)目的片段连接pET28a表达载体并转化至BL21(DE3)感受态
4.5μL的目的片段(胶回收产物)加0.5μL的pET-28a表达载体在16℃水浴锅过夜连接,将连接产物普转转入BL21(DE3)感受态中,涂板于带有Kana抗性(50mg/mL)的固体LB平板上;选取平板上大小适中的单菌落,挑菌至液体LB中培养,提取质粒,双酶切测定,送样质粒测序。
1.2.12p14.5蛋白的纯化
a.接菌BL21-pET-28a-p14.5,从-80℃取出保存的菌在5mL的Kana抗性的LB培养基中培养8h;b.转接,800mLKana抗性的LB液体培养基加入a中菌液10mL,培养至od600=0.6-0.8;c.诱导,菌液中加入IPTG(200mg/mL)诱导剂250μL诱导表达4h;d.离心,将诱导后菌液1000rpm离心10min,5mL PBS重悬菌体沉淀;e.超声破碎后,10000rpm,4℃条件下离心20min,收集沉淀(收集部分上清留作检测);f.用2M尿素PBS洗一次、用4M尿素PBS洗一次、用8M尿素PBS洗一次,用0.22μm除菌过滤器过滤;
g.离心取上清液,沸水煮样处理10min;
h.跑western,将胶用0.25M Kcl处理,切胶至碎块状,用PBS浸泡7-8h;离心,吸取上清。
1.3结果
1.质粒pLP-S-p14.5,pLP-S-p14.5-IL-33以及pLP-S-CTA1-p14.5-D-D的核苷酸序列分别如SEQ ID NO.3-5所示;
2.质粒pLP-S-p14.5,pLP-S-p14.5-IL-33以及pLP-S-CTA1-p14.5-D-D的氨基酸序列分别如SEQ ID NO.6-8所示;
3.质粒pLP-S-p14.5,pLP-S-p14.5-IL-33以及pLP-S-CTA1-p14.5-D-D鉴定
将合成的质粒用SalⅠ和XhoⅠ双酶切分别得到载体和目的片段,如图1、图2、图3所示。
4.质粒图谱
成功构建好的NC8-pLP-S-p14.5如图4所示,NC8-pLP-S-p14.5-IL-33-Mus如图5所示,NC8-pLP-S-CTA1-p14.5-D-D如图6所示。
5.p14.5扩增PCR结果
P14.5基因PCR扩增产物核酸电泳验证,扩增基因片段大小为402,如图7所示;
6.构建成功的BL21-pET-28a-p14.5酶切鉴定结果
将构建好的BL21-pET-28a-p14.5取质粒用XhoⅠ和NcoⅠ进行双酶切,结果与预期相符;如图8所示。
7生长曲线测定以及最佳稀释倍数菌落计数
NC8-pLP-S、NC8-pLP-S-P14.5、NC8-pLP-S-P14.5-IL-33-Mus、NC8-pLP-S-CTA1-P14.5-D-D的生长曲线测定结果(12h)以及多次菌落滴板计数测定结果进行滴板计数结果选择12h为最佳饲喂时间点(即加入诱导剂SppIP后8h)。菌株生长曲线如图9所示。
实施例2
重组植物乳杆菌的动物实验
实施例1已经成功构建了三株重组植物乳杆菌并且体外锚定表达成功,该实施例选择小鼠C57BL/6作为实验动物进行初步动物实验,通过口服饲喂的方式免疫小鼠,通过流式细胞术,淋巴细胞增殖试验,ELISA等检测免疫小鼠的体液免疫,细胞免疫,粘膜免疫等方面的变化,从而研究新功能型乳酸菌对于免疫动物的免疫力提升作用。
2.1材料
2.1.1菌株
NC8-pLP-S-p14.5、NC8-pLP-S-p14.5-IL-33-Mus、NC8-pLP-S-CTA1-p14.5-D-D由实施例1获得;
2.1.2实验动物
5-6周龄的SPF级别的雌性C57BL/6小鼠50只购自北京华阜康生物科技股份有限公司;于吉林农业大学创新大楼SPF级别饲养室饲养,适应环境1周后进行试验。
2.1.3主要试剂
小鼠耳标器、剪刀、镊子、1.5mL EP管、灌胃针小号、眼球采血管、PBS、24孔细胞培养板、96孔酶标ELISA检测板、细胞1640培养液均购自北京全式金生物公司;流式抗体CD3、CD4、CD8、CD11、CD80、CD86、B220、IgA、IFN-γ、IL-4均购自BD公司。
2.1.4主要仪器
低温超速离心机(Eppendorf 5810R);SW-CJ-2FD型单面双人超净工作台(上海博迅);HRLM-80型全自动高压灭菌锅、BCD-649WDCE型冰箱(海尔公司);G70D20CN1P-D2(S0)20L型微波炉(中国格兰仕);Innova 40R型恒温摇床(美国NBS).
2.2方法
2.2.1实验分组
将50只小鼠随机分成5组,每组10只。每组分别饲喂PBS,NC8-pLP-S,NC8-pLP-S-p14.5,NC8-pLP-S-p14.5-IL-33-Mus,喂NC8-pLP-S-CTA1-p14.5-D-D,在每只小鼠右耳上标号,每组标10个自然数字号码。
2.2.2乳酸菌饲喂
由实施例1的1.3.7已知最佳饲喂条件,同方法进行接种培养,通过口服的方式将新功能型重组植物乳杆菌对小鼠进行饲喂。
2.2.3眼球静脉采血
用静脉取血针找好小鼠眼角位置,轻柔旋转进针,待出血后引流入1.5mL EP管中,取0.2mL血液即可。
2.2.4粪便收集
每三天进行一次粪便的收集。
2.2.5单细胞悬液制备方法
在超净台中,用眼科剪,眼科镊(已高压灭菌)剥离肠系膜淋巴结(MLN)、派依氏集合淋巴结(PP)和脾脏,将多余的脂肪摘除。将叠好的200目无菌滤网后放置于无菌小平皿中,加入1mL RPMI-1640培养液。将脾放入滤网中,用无菌1mL注射器末端轻轻研磨,直到研磨充分后,将液体吸到1.5mL EP管中,放入预冷的离心机中,4℃,2000rpm,离心5min。然后弃上清,加0.5mL红细胞裂解液,冰上裂解3min后加0.5mL PBS混匀静止2min。2000rpm,4℃离心5min,弃上清。1mL PBS洗1遍弃上清加1mL完全培养基。稀释100倍后细胞计数板计数。MLN、PP结以同样的方式处理,但不需要用红细胞裂解液裂解。处理后,稀释20倍进行计数。
2.2.6抗体染色
染色方法详见流式抗体染色说明书。
2.2.7流式上机
2.2.8ELISA检测
检测粪便中分泌型SIgA和血清中IgG。
(1)包被:在96孔培养板的每个样品孔包被纯化后的P14.5抗原(浓度为1μg/mL),每孔100μL,4℃条件下静置过夜;(2)洗涤:每个样品孔加含0.05%吐温-20的PBS室温下洗涤,300μL/孔,每5min洗涤一次,共三次;(3)封闭:每个样品孔加入含有1%BSA的PBS,4℃封闭过夜,150μL/孔;(4)洗涤:重复步骤2;(5)一抗:加入待检测的样品(粪便与血清处理后的样品),封膜;(6)洗涤:重复步骤2;(7)二抗:二抗为稀释后的抗鼠SIgA与抗鼠的IgG,封膜;(8)洗涤:重复步骤2;(9)底物显色,加入显色液100μL/孔,37℃条件下避光反应1h;(10)终止反应:添加终止液10%H2SO4,50μL/孔;(11)酶标仪检测结果,检测波长为450nm,保留数据;(12)分析结果。
2.2.9CCK-8比色法检测T淋巴细胞增殖96孔板
1、制备细胞悬液:细胞计数;2、接种到96孔板中:根据合适的铺板细胞数(约1-2×104),每孔约100ul细胞悬液,同样的样本可做4-6个重复;3、37℃培养箱中培养:细胞接种后贴壁大约需要培养4小时,如果不需要贴壁,这步可以省去;4、加入10μL CCK8:由于每孔加入CCK8量比较少,有可能因试剂沾在孔壁上而带来误差,建议将枪头浸入培养液中加入且在加完试剂后轻轻敲击培养板以帮助混匀。或者直接配置含10%CCK8的培养基(现用现配),以换液的形式加入;5、培养0.5-4小时:细胞种类不同,形成的Formazan的量也不一样。如果显色不够的话,可以继续培养,以确认最佳条件(建议预实验先摸清楚时间点)。特别是血液细胞形成的Formazan很少,需要较长的显色时间(5-6小时);6、测定450nm吸光度:建议采用双波长进行测定,检测波长450-490nm,参比波长600-650nm。
2.3结果
2.3.1流式细胞术检测结果
2.3.1.1脾脏淋巴细胞中CD3+CD4+T细胞中IL-4、IFN-γ和CD3+CD8+T细胞中IL-4、IFN-γ的含量显著上升
脾脏是主要的外周免疫器官,是T细胞和B细胞定居的场所,同时也是体内产生抗体的主要器官,在机体的防御、免疫应答中具有重要地位。CD3、CD4、CD8是淋巴细胞的表面标志,可以标记细胞毒性T细胞和辅助性T细胞。IL-IL-4主要由活化T细胞产生,对于B细胞、T细胞、肥大细胞、巨噬细胞和造细胞都有免疫调节作用,IFN-γ具有抗病毒,抗肿瘤和免疫调控的作用,同时可以促进NK细胞活性,促进抗原呈递和提高巨噬细胞溶酶体活性。
重组植物乳杆菌增强了对机体的免疫效果,脾脏淋巴细胞中CD3+CD4+T细胞中IL-4、IFN-γ和CD3+CD8+T细胞中IL-4、IFN-γ的含量显著上升;如图10-13所示。
2.3.1.2派依氏集合淋巴结(PP)细胞中11C+80+和11C+86+含量的变化
派尔集合淋巴结(Peyer patch),又称peyer斑、派尔斑、PP结、肠道集合淋巴结,Peyer patch等,是肠黏膜免疫系统的重要组成部分,是小肠粘膜内的一组淋巴滤泡。
淋巴滤泡由B细胞和T细胞(CD4为主)组成,在其表面覆盖着一层微皱褶细胞,又称M细胞。它能识别胃肠道内呈现的许多抗原,主要吞噬病毒和肠道病原菌,递呈吞入的肠腔内抗原转交给免疫细胞,免疫细胞可对致病抗原加工、转运、呈递。在此过程中被激活的免疫细胞经过循环归巢的过程回到肠黏膜固有层,成为分泌IgA为主的浆细胞和效应T细胞参与肠道局部免疫反应。主要存在回肠部位。
CD80是CD86活化T淋巴细胞时的协同刺激因子,在自身免疫监控、体液免疫应答及移植反应中发挥重要作用。CD86与诱导剂CD28和抑制剂CTLA4相互作用,是诱导T淋巴细胞增殖及产生IL-2的主要协同因子。
重组乳杆菌显著增强PP中11C+80+和11C+86+的表达;如图14和15所示。
2.3.1.3派依氏集合淋巴结(PP)细胞中B220+和IgA+含量的变化
PP是宿主小肠的次级淋巴器官,聚集了B细胞、M细胞等各种细胞,其中M细胞在抗原的摄取过程中发挥了重要的作用。PP存在于从粘膜到粘膜下层,B细胞位于生发中心,T细胞则在其周围。其中PP承担监控肠腔抗原的粘膜免疫反应的任务,B220和IgA是B细胞活化标志,所以PP中活化B细胞的水平是评价机体免疫的重要指标。
重组乳杆菌显著增强PP中B220+IgA+的表达;如图7所示。
2.4实施例1成功构建了NC8-pLP-S-p14.5,NC8-pLP-S-p14.5-IL-33-Mus和NC8-pLP-S-CTA1-p14.5-D-D,并验证了它们在体外的成功表达。由于乳酸菌可以在小鼠肠道内定殖,并能在肠道环境下可以刺激乳酸菌表达p54蛋白以及p54-IL-33、CTA1-p14.5-D-D融合蛋白,机体T细胞识别p54蛋白以及p54-IL-21融合蛋白并产生特异性的抗体。用小鼠进行初步动物试验,结果可见饲喂重组植物乳杆菌对于小鼠的免疫效果具有一定的提升作用。
由流式细胞术实验可见饲喂重组植物乳杆菌组小鼠的PP结淋巴细胞中B220和IgA的分泌的量以及11C+CD80和11C+CD86的含量大于对照组;脾脏中CD4+IFN-γ+CD4+IL-4和的CD8+IFN-γ+CD8+IL-4表达量也有显著的增强。
以上一系列实验结果都表明了当饲喂新功能型重组植物乳杆菌后,小鼠的免疫力有显著的提高。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (1)
1.一种抗病毒产品,其特征在于,所述抗病毒产品包括重组植物乳杆菌;
所述重组植物乳杆菌转化有非洲猪瘟病毒抗原蛋白重组表达载体,所述重组植物乳杆菌为:NC8-pLP-S-CTA1-p14.5-D-D;
所述非洲猪瘟病毒抗原蛋白重组表达载体中包括非洲猪瘟病毒编码p14.5蛋白的E120R基因序列;所述重组表达载体为pLP-S-CTA1-p14.5-D-D;
CTA1-p14.5-D-D的核苷酸序列如SEQ ID NO.5所示;
CTA1-p14.5-D-D编码氨基酸序列如SEQ ID NO.8所示的蛋白;
所述病毒为非洲猪瘟病毒;
所述产品为猪疫苗,所述疫苗剂型为口服冻干粉剂;
所述重组植物乳杆菌的构建方法包括:
S1、将编码p14.5蛋白的E120R基因序列和佐剂CTA1-DD基因片段插入以erm为筛选标记单锚定表达载体pLP-S中,构建重组表达载体pLP-S-CTA1-p14.5-D-D;
S2、获得植物乳杆菌NC8感受态;
S3、将步骤S1的重组表达载体电转化至S2得到的植物乳杆菌NC8感受态中即得。
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