CN114645052B - 一种全脑过表达核易位人源α-突触核蛋白转基因鼠的高效构建方法 - Google Patents
一种全脑过表达核易位人源α-突触核蛋白转基因鼠的高效构建方法 Download PDFInfo
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Abstract
本发明公开一种全脑过表达核易位人源α‑突触核蛋白转基因鼠的高效构建方法本发明引入3个重复序列的核易位信号肽，通过PCR方法获取人源α‑Syn‑3*NLS编码序列，以重组腺相关病毒rAAV过表达病毒载体pAAV‑IRES‑hrGFP为基本骨架，通过双酶切位点SalⅠ和Xhol，将目的序列α‑Syn‑3*NLS构建入载体中，进行病毒包装得到过表达核易位α‑Syn的rAAV病毒，对乳鼠采用脑立体汉密顿微量注射针通过定位方法侧脑室注射该病毒，获得全脑过表达核易位人源α‑突触核蛋白转基因鼠，以解决现有过表达α‑突触核蛋白转基因小鼠制作，以及α‑突触核蛋白缺乏核输入信号，无法在核内过表达的问题。
Description
技术领域
本发明涉及生物技术领域，具体涉及全脑过表达核易位人源α- 突触核蛋白转基因鼠的高效构建方法。
背景技术
1997年，Spllantini等人报道，SNCA编码的α-突触核蛋白(alpha synuclein,α-Syn)是帕金森病(Parkinson’s disease,PD)主要病理—路易小体的主要组成成分，随后又有研究报道SNCA基因的突变型(A30P,E46K,H50Q,G51D,A53E,A53T)和其二倍体、三倍体均能诱导PD的发生，表明SNCA基因在PD的病理进程中发挥着重要的作用。α-Syn因其最初发现存在于突触前神经末梢和核膜周围而得名，随后的研究证实其在细胞系、果蝇、转基因小鼠及阿尔茨海默症和 PD患者脑组织中均存在核定位的现象。核易位的α-Syn被证实能与 DNA相互作用，引起DNA损伤，引发神经毒性，造成神经元的死亡。表明核易位的α-Syn参与PD的发生发展过程。因此，基于核易位α-Syn细胞模型和动物模型的建立用于研究PD病理机制以及药物筛选成为亟待解决的关键问题。
相比较传统的毒素模型和转基因动物模型，采用定位注射重组腺相关病毒(rAAV)递送的α-Syn构建α-Syn局部过表达动物模型，能初步复现PD的原发性运动障碍及部分多巴胺鞥神经元损伤，因此备受研究者的关注。然而，由于其基因组不整合的特点只能实现局部过表达。
现有技术中，α-突触核蛋白缺乏核输入信号，还无法在核内过表达。
发明内容
本发明的目的在于提供一种全脑过表达核易位人源α-突触核蛋白转基因鼠的高效构建方法，以解决现有技术采用重组腺相关病毒 (rAAV)递送基因时，由于不整合基因组而只能实现局部过表达的问题，以及解决α-突触核蛋白缺乏核输入信号，无法在核内过表达的问题。
本发明通过下列技术方案实现本发明目的：一种全脑过表达核易位人源α-突触核蛋白转基因鼠的高效构建方法，经过下列各步骤：
第一步：通过引物设计，引入3个重复序列的核易位信号肽 (3*NLS)，通过PCR方法获取人源核易位α-Syn(α-Syn-3*NLS) 序列；所述核易位信号肽序列为CCAAAAAAGAGAAAGGTA；
第二步：以AAV过表达病毒载体pAAV-IRES-hrGFP为基本骨架，通过双酶切位点SalⅠ和Xhol将目的序列α-Syn-3*NLS构建入载体中，通过DNA测序方法确定基因序列准确插入，命名重组质粒为 AAV-α-Syn-3*NLS；
第三步：采用293T对AAV-α-Syn-3*NLS质粒进行病毒包装，得到过表达核易位α-Syn的rAAV载体；包装病毒感染SH-SY5Y细胞后，采用免疫荧光方法和Western blot方法对病毒包装进行验证；验证表达和包装方法无误的过表达核易位α-Syn的rAAV病毒进行浓缩和病毒滴度测定；
第四步：对乳鼠侧脑室定位注射过表达核易位α-Syn的rAAV载体，滴度为1×1013vector genome(vg)/ml，获得全脑过表达核易位人源α-突触核蛋白转基因鼠。
采用免疫荧光染色方法和Western blot方法检测各脑区的核易位α-Syn表达情况，验证全脑过表达核易位人源α-突触核蛋白转基因小鼠构建是否成功。
工作原理：本发明，基于目前研究核易位α-Syn在PD中的功能作用，通过PCR方法引入3个重复的核易位信号序列(NLS)，获取编码核易位的α-Syn(α-Syn-3*NLS)的基因序列。并通过载体构建获得过表达核易位的α-Syn的rAAV病毒载体，提供一种增强型核易位α-Syn载体的构建方法。通过病毒包装，将1013vg/ml的病毒颗粒通过侧脑室定位注射乳鼠，注射后14d，可成功获得全脑过表达核易位的α-突触核蛋白的转基因小鼠，为开展研究核易位的α-突触核蛋白与PD的病理机制提供研究工具。有益效果：
(1)本发明过向胎鼠或乳鼠脑室内注射rAAV来实现整个中枢神经系统的基因过表达，实现了核易位α-Syn的表达，通过免疫荧光确认了α-Syn的细胞定位，明显定位为核内，可通过此过表达载体构建核易位α-Syn细胞模型和动物模型。
(2)本发明通过侧脑室脑立体定位的方法，实现了全脑过表达核易位α-Syn的转基因小鼠模型。
附图说明
图1为本发明使用的rAAV载体图谱；
图2为本发明采用荧光显微镜10倍镜下观察包装的rAAV感染 293T细胞72h后的荧光效果；
图3为本发明采用Western blot方法检测核易位α-Syn的rAAV 表达情况；
图4为本发明采用免疫荧光方法检测核易位α-Syn在细胞中的定位情况；
图5为本发明定位注射示意图；
图6为本发明免疫荧光方法检测定位侧脑室注射乳AAV-α-Syn -3*NLS两周后，小鼠全脑切片中α-Syn的表达情况；
图7为本发明免疫荧光方法检测定位注射乳鼠侧脑室过表达核易位α-Syn的rAAV两周后小鼠中脑中核易位α-Syn的表达情况检测；
图8为本发明Western blot方法检测过表达核易位α-Syn转基因鼠各脑区中α-Syn的表达情况；
图9为本发明技术路线。
具体实施方式
下面结合附图和实施例对本发明做进一步说明。
第一步：核易位α-Syn编码序列的获取：
通过引物设计引入3个重复的核易位序列(NLS)，获取核易位α-Syn序列，扩增引物序列为：
上游引物
AAGTCGACGCCACCATGGATGTATTCAT
下游引物
AACTCGAGTTATACCTTTCTCTTTTTTGGTACCTTTCTCTTTTTTGGTACCTTTCTCTTTTTTGGGGCTTCAGG
下划线处表示酶切位点。PCR扩增条件为：94℃预变性5min， 94℃变性30s，55℃退火30s，72℃延伸1min，循环35次，最后72℃延伸10min。扩增后通过1.5％的琼脂糖凝胶电泳检测，目的片段大小约为497bp。
下面是过表达核易位α-Syn的编码序列：
ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTG GCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGA CAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTG CATGGTGTGGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGT TGGAGGAGCAGTGGTGACGGGTGTGACAGCAGTAGCCCAGAAGACAGTG GAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACC AGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGAT ATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGG TATCAAGACTACGAACCTGAAGCCCCAAAAAAGAGAAAGGTACC AAAAAAGAGAAAGGTA CCAAAAAAGAGAAAGGTATAA
下划线部分为3个重复的核易位信号肽序列(3*NLS)，核易位信号肽序列为CCAAAAAAGAGAAAGGTA。
第二步：过表达核易位α-Syn的rAAV载体的构建
按照试剂盒说明书，分别采用SalⅠ和Xhol限制性内切酶酶切载体pAAV-IRES-hrGFP和目的片段α-Syn-3*NLS，并按试剂盒方法回收产物。通过T4连接酶，将目的片段构建入pAAV-IRES-hrGFP载体中，转化宿主菌DH5α，铺板于终浓度为100μg/ml的LB固体平板上。对阳性克隆子抽提质粒后进行双酶切和DNA测序鉴定，鉴定正确的阳性质粒命名为AAV-α-Syn-3*NLS。
第三步：过表达核易位α-Syn的rAAV病毒包装
采用对数生长期的293T细胞传于15cm的培养皿，待细胞融合度为80％左右进行病毒包装。具体地：准备DNA mix管和转染试剂管。DNA mix管：目的基因质粒(7.5μg):辅助质粒(pRC9:Helper) ＝1:1，混匀于500μl Opti-MEM培养基中。转染试剂管：20μl rAAV 转染试剂混匀于500μl Opti-MEM培养基中。将DNA mix管中的试剂缓慢加入转染试剂管中，轻轻混匀后室温静置15min。将混匀试剂缓慢滴加到更换为灭活血清配制的完全培养基的293T细胞皿中，轻轻晃动后放回细胞培养箱继续培养。培养8h后更换为新鲜的灭活血清配制的完全培养基继续培养到72h，采用荧光显微镜观察细胞荧光情况。
图2为采用荧光显微镜10倍镜下观察包装的rAAV感染293T细胞72h后的荧光效果。因质粒骨架上携带EGFP荧光标签，包装成功的病毒感染细胞后可以表达EGFP从而发出绿色荧光。结果显示过表达核易位α-Syn的rAAV包装成功。
图3为Western blot方法检测核易位α-Syn的rAAV表达情况。对感染过表达核易位α-Syn的rAAV成功的293T细胞裂解后提取蛋白采用anti-α-Syn抗体进行检测，GAPDH作为内参蛋白。检测结果显示，相比较空载体对照组，过表达核易位α-Syn的rAAV实验组在 20KD左右处有一特异性条带，证实核易位α-Syn在rAAV中表达成功。
第四步：过表达核易位α-Syn的rAAV病毒浓缩
按商品化方法进行浓缩。
第五步：过表达核易位α-Syn病毒包装成功验证
取浓缩病毒加入铺于6孔板/放置细胞爬片的24空板的293T细胞中，加入终浓度为8μg/ml的助感染试剂polybrene,8h后更换为新鲜的培养基。继续培养至72h后，荧光显微镜观察细胞绿色荧光情况，细胞具有绿色荧光证实病毒感染成功，进一步对6孔板的细胞提取蛋白后采用Western blot方法检测α-Syn的表达情况。同时24孔爬片进行进过4％多聚甲醛固定后采用anti-SNCA抗体通过免疫荧光方法检测α-Syn在细胞中的定位情况。
图4为采用免疫荧光方法检测核易位α-Syn在细胞中的定位情况。对感染过表达核易位α-Syn的AAV成功的293T细胞通过多聚甲醛固定后，采用人源α-Syn特异性一抗anti-α-Syn进行检测，红色荧光标记的为α-Syn，DAPI标记细胞核。如图5Merged所示，通过激光共聚焦显微镜拍摄显示：α-Syn蛋白明显定位于细胞核内，说明过表达核易位α突触核蛋白的rAAV成功，实现了α-Syn在核内的定位表达。命名为AAV-α-Syn-3*NLS。
第六步：侧脑室注射乳鼠AAV-α-Syn-3*NLS构建核易位α-Syn 转基因鼠
刚出生的乳鼠进行冰麻，把固绿和AAV-α-Syn-3*NLS进行1:2 体积比例混合均匀，AAV-α-Syn-3*NLS的滴度约为1×1013vg/ml，采用规格为2.5μl的汉密顿注射针(注射器货号7632-01，针头货号 7803-05)进行定位注射，注射位点为：lambda点与小鼠眼睛连线的2/5处(靠近lambda点)，注射深度为3mm，注射时间为1min，注射结束后留针30s。每侧注射携带EGFP(空质粒对照)和AAV-α -Syn-3*NLS的与固绿的混合液1μl。剪脚趾作好标记后，放回母鼠身边继续给予充足的食物和水源饲养，并于笼上标记注射时间及注射的小鼠数量。
图5为定位注射示意图
A图；定位注射进针位点示意图，圆圈位置指示注射进针位点。
B图：注射位置示意图。
第七步：转基因小鼠检测
注射14d后，转基因小鼠一方面通过4％多聚甲醛灌注后取脑，固定48h后，经过脱水包埋，制作石蜡切片。采用anti-α-Syn抗体进行免疫荧光染色方法检测核易位α-Syn在小鼠全脑中的表达。另一方面小鼠通过处死取脑，分离各脑区分别加入蛋白裂解液，通过超声匀浆离心，提取蛋白后，采用Western blot检测核易位α-Syn的表达。
图6为免疫荧光方法检测定位侧脑室注射乳鼠AAV-α-Syn -3*NLS两周后，小鼠全脑切片中α-Syn的表达情况。相比较空质粒对照组，荧光结果显示核易位α-Syn在转基因小鼠的全脑区均高效表达。图7为免疫荧光方法检测定位注射乳鼠侧脑室过表达核易位α -Syn的r AAV两周后小鼠中脑中核易位α-Syn的表达情况检测。分别用anti-α-Syn抗体，anti-Neurn抗体标记神经元，采用DAPI标记细胞核。从图中Merged图可明显观察到α-Syn在神经元细胞核中高效表达。
图8为Western blot方法检测过表达核易位α-Syn转基因鼠各脑区中α-Syn的表达情况。采用人源α-Syn特异性一抗anti-α-Syn对转基因小鼠各脑区蛋白裂解液进行检测，相比较空质粒对照组，过表达核易位α-Syn实验组在20KD作用处明显有特异性条带，证实核易位α -Syn在小鼠各脑区成功表达。
序列表
<110> 中国医学科学院医学生物学研究所
<120> 一种全脑过表达核易位人源α-突触核蛋白转基因鼠的高效构建方法
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Claims (1)
1.一种全脑过表达核易位人源α-突触核蛋白转基因鼠的高效构建方法，其特征在于：包括以下步骤：
（1）通过引物设计，引入3个重复序列的核易位信号肽，通过PCR方法获取人源核易位α-Syn，即α-Syn-3*NLS序列；所述核易位信号肽序列为CCAAAAAAGAGAAAGGTA；
（2）以AAV过表达病毒载体pAAV-IRES-hrGFP为基本骨架，通过双酶切位点SalⅠ和Xhol将目的序列α-Syn-3*NLS构建入载体中，通过DNA测序方法确定基因序列准确插入，命名重组质粒为AAV-α-Syn-3*NLS；
（3）采用293T对AAV-α-Syn-3*NLS质粒进行病毒包装得到过表达核易位α-Syn的rAAV载体；
（4）对乳鼠采用侧脑室定位注射过表达核易位α-Syn的rAAV载体，滴度为1×1013 vg/ml，获得全脑过表达核易位人源α-突触核蛋白转基因鼠。
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