CN112143755A - 一种人甲状旁腺激素真核表达重组质粒载体及其构建方法 - Google Patents
一种人甲状旁腺激素真核表达重组质粒载体及其构建方法 Download PDFInfo
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- CN112143755A CN112143755A CN202011114208.8A CN202011114208A CN112143755A CN 112143755 A CN112143755 A CN 112143755A CN 202011114208 A CN202011114208 A CN 202011114208A CN 112143755 A CN112143755 A CN 112143755A
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Abstract
本发明涉及基因药物领域,具体提供了一种人甲状旁腺激素真核表达重组质粒载体及其构建方法。该构建方法包括如下步骤:人工合成甲状旁腺激素(hPTH1‑84aa)基因(SEQ ID NO.1),经PCR扩增获得甲状旁腺激素基因片段,用限制性内切酶BamHI和XhoI双酶切PCR产物后,与经相同酶切的真核表达载体质粒PHY‑810连接,将连接好的表达载体转化感受态细菌,涂板筛选得到人PTH基因真核表达重组质粒载体(图1:PHY‑PTH)。本发明所构建的真核表达重组质粒载体使细胞高表达PTH,可用于针对甲状旁腺功能减退症和骨质疏松的基因治疗。
Description
技术领域
本发明涉及基因药物领域,特别涉及针对甲状旁腺功能减退基因治疗的重组质粒载体构建。
背景技术
甲状旁腺素是由人甲状旁腺分泌的活性多肽类激素,其主要生理功能是通过调节骨细胞和肾细胞维持血钙平衡,刺激肾对钙的重吸收、磷的分泌和骨的重建,是调节钙磷代谢的重要肽类激素。
甲状旁腺机能减退症是由于疾病、衰老和手术损伤引起的甲状旁腺激素分泌不足所导致的一种钙磷代谢调节障碍性疾病。疾病、衰老导致的甲状旁腺机能减退症以脱钙、骨骼再生障碍、骨质疏松为特征;手术损伤引起的甲状旁腺机能减退症又分为暂时型和永久型,主要临床特征以低血钙导致的低钙抽搐和心血管系统功能障碍为特征。
由于疾病、衰老导致的甲状旁腺机能减退症,多见于老年化、孕产妇、更年期妇女、长期使用糖皮质激素的自免疾病患者和肿瘤患者。临床上表现为骨质疏松,随着老年化趋势凸显,发病率持续攀升,中国目前的骨质疏松人数达到2.5亿。 手术损伤引起的甲状旁腺(PTG)损伤是急性继发性甲状旁腺机能减退症主要原因。我国每年由于甲状旁腺损伤导致甲旁功能减退达到50万以上,到2019年,中国永久性甲状旁腺损伤累计人数超过500万。
针对甲状旁腺机能减退症,目前除了补钙等对症替代疗法,尚无有效对策。甲状旁腺激素补充和替代疗法成为从源头上阻断疾病发生发展的主要策略。随着基因工程技术发展,美国礼来制药公司首先开发出针对提升甲状旁腺激素水平的基因工程产品-重组人甲状旁腺激素(PTH1-34aa)注射液(特立帕肽注射液),2002年获得美国FDA批准上市,2011 年3月份,被中国国家食品药品监督管理局批准在中国上市,用于治疗甲旁低和钙磷代谢障碍导致的严重骨质疏松。
但现用的重组人甲状旁腺激素制剂存在不足之处,影响力临床使用。首先重组PTH在体内在体内代谢快,很不稳定,需每日定时用药维持血药浓度,因此增加了用药难度和影响疗效;第二是毒副作用,用药后可出现眩晕,肌肉酸痛,呼吸困难,恶心呕吐;更大的限制是本品治疗最长时间不能超过24个月,而且病人终生只可以介绍一次最长24个月的治疗。第三是制备成本高:由于PTH表达产物含量很低,分离纯化困难,导致制备成本高,药物昂贵,限制了规模化使用。
人们不断在重组PTH制备工艺和产品结构上进行改进和优化,设法克服和弥补现用重组PTH制剂的缺陷,所以目前国内外针对甲旁素的基因工程技术专利主要集中在优化重组人甲状旁腺激素产品性能和提升产量等方面。如:范文超于2012年申请了发明专利【人甲状旁腺激素1-34 的制备方法】(CN 102304518 A),该发明将编码人甲状旁腺激素的基因与硫氧还蛋白标签融合表达,并进一步构建高表达PTH工程菌; 丁邦等发明专利【重组人甲状旁腺激素PTH1-34 的制备方法】(CN 1807456 B)提供了含重组人甲状旁腺激素的Trx-PTH1-34 融合蛋白,赵彬等的发明专利(CN 1256431C)构建了基因重组表达载体和重组人PTH的制备方法。从专利查询平台检索已申报的相关PTH基因工程产品专利,绝大部分是关于重组甲状旁腺激素制备的专利,其余部分是关于甲状旁腺激素制剂等相关专利,但是到目前为止,未能从根本上突破重组PTH制剂的局限性。
基因治疗是根本上解决甲旁腺功能减退症的有效途径。通过基因治疗使目前的体外补充PTH方法转变为体内表达PTH,避免目前临床替代治疗所替面临的毒副作用和局限性。基于这一目标,本发明构建PTH重组质粒表达载体用于针对甲状旁腺功能减退症的基因治疗。重组质粒表达载体作为一类非病毒载体,有着安全性高,制备简单、方便快捷等优点。经专利检索目前尚未查到构建PTH重组质粒表达载体进行基因治疗的专利。通过构建PTH重组表达质粒作为基因治疗载体,开创了甲旁功能减退治疗的新途径。
发明内容
1. 发明目的。
本发明的第一个目的在于提供一种人甲状旁腺素真核表达重组质粒载体,用于针对甲状旁腺功能减退的基因治疗。
本发明的另外一个目的是提供一种人甲状旁腺素真核表达重组质粒载体的构建方法。
2. 在本发明的第一个方面,提供了一种人甲状旁腺激素重组质粒真核表达载体。其特征在于,它包括以下原件。
(1)人甲状旁腺激素PTH(1-84aa)基因序列:SEQ ID No 1序列,包含intact PTH序列完整结构。
(2)质粒载体为PHY-810,载体原件:CMV-MCS-3xFlag-SV40-Neo,质粒大小:5491bp。
(3)甲状旁腺激素基因克隆位点位于BamHI—XhoI。
(4)3xFlag 位于PTH(1-84aa)基因的C端。
(5)其结构特征在于利用了载体本身的flag-tag,在不增加重组载体bp数量的前提下,避开N端功能区,在C端连接flag-tag,使PTH与3xFlag融合表达,提高重组蛋白质的产量、增强重组蛋白质的可溶性和稳定性。
3. 在本发明的第二方面,提供了一种过表达人甲状旁腺素的重组质粒真核表达载体的构建方法,通过以下步骤实现。
(1)人工合成人甲状旁腺激素(PTH1-84aa)的基因cDNA(SEQ ID NO. 1)。
(2)以所合成的PTH基因为模板,设计引物,其特征在于基于目的基因序列的引物设计,分别在上游引物的5’端插入一个BamHI酶切位点,在下游引物的5’端插入一个XhoI酶切位点:p1(BamH I):CTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGGGTGTGTGTATGTG;p2(XhoI):CATGGTCTTTGTAGTCCTCGAGCTGGGATTTAGCTTTAGTTAATACAT,将设计引物委托合成。
(3)经PCR扩增出甲状旁腺激素基因片段:其特征在于PCR扩增条件为:95℃预变性3min,然后95℃15s,60℃15s,72℃30s条件下扩增30个循环,最后72℃延伸5min。
(4)扩增的目的基因片段用BamHI和XhoI双酶切后,与经相同酶切的载体PHY-810连接,将PTH目的基因连入酶切后的PHY-810载体上. 其特征在于。
a. 所述目的基因扩增产物与PHY-810载体的体积比=(载体长度×0.02)/(目的片段长度X0.04)。
b. 目的基因片段插入到PHY-810载体多克隆区,酶切位点方位 BamH I-Xho I,3xFlag 标签位置C 端。重组质粒大小为5888bp。
(5)将连接好的表达载体转化感受态细菌, 然后涂板筛选重组质粒。
(6)筛选出质粒首先经酶切后电泳和PCR鉴定,再进行测序鉴定,得到含人PTH基因的重组质粒真核表达载PHY-PTH,其特征在于测序结果报告序列为:SEQ ID NO.2。
4. 表达验证:本发明提供的人甲状旁腺素重组质粒真核表达载体, 其表达293T细胞上清中检测到PTH高表达。
5. 有益效果:本发明所提供的PHY-PTH作为基因治疗载体,可以使甲状旁腺外细胞表达PTH,从而起到替代甲状旁腺分泌PTH的治疗作用。重组质粒表达载体PHY-PTH作为一类非病毒载体、有着安全性高、性能稳定、制备简单、方便快捷、易于规模化标准化生产、易于储存和运输等优点。
附图说明
为了更清楚地说明本发明实施例的技术方案,以下附图是对实施例中所述内容的图解说明,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对本发明范围的限定。
图1. 摘要附图。
图2. 构建PTH重组质粒表达载体技术路线示意图。
图3. PTH重组质粒载体酶切鉴定。
图4. PTH重组质粒载PCR鉴定。
图5. PTH重组质粒载体测序结果文件。
图6. FLAG表达验证:WEST BLOT 检测到FLAG在PTH重组质粒转染的293T细胞中成功表达。
图7. PTH表达验证(免疫化学发光法):在PHY-PTH转染的293T细胞上清中检测到PTH高表达。
具体实施方式
下面结合具体实例对本发明的方法进行详细描述和说明。其内容是对本发明的解释而非限定本发明的保护范围。
实施例一: 人甲状旁腺激素真核表达重组质粒载体构建(图2)。
1. 目的基因的合成、扩增和酶切。
(1)人工合成人甲状旁腺激素(PTH1-84aa)的cDNA(SEQ ID NO. 1)。
(2)以所合成的PTH基因为模板,设计引物,其特征在于基于目的基因序列的引物设计,分别在上游引物的5’端插入一个BamHI酶切位点,在下游引物的5’端插入一个XhoI酶切位点:p1(BamH I):CTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGGGTGTGTGTATGTG;p2(XhoI):CATGGTCTTTGTAGTCCTCGAGCTGGGATTTAGCTTTAGTTAATACAT。将设计好的引物序列送生工进行合成。将合成的引物稀释成终浓度为10µmol/L的储藏液。
(3)利用稀释的引物及SEQ ID NO. 1模板进行PCR扩增。体系如下:
模板 1-2 µg
引物1 2 μL
引物2 2 μL
PCR mix 25 μL
ddH2O 补足50μL
将上述材料加入薄壁管内混匀并点离后放入PCR仪内,PCR条件:PCR条件:预变性94度,变性98度,2min,变性98度,10s,退火62度,30s,延伸68度,3min,30个循环,最后延伸68度,5min。
(4)PCR结束后进行琼脂糖凝胶电泳,并回收目的基因。
(5)将回收后的目的基因用BamHI(内切酶1)和XhoI(内切酶2)双酶切,
酶切体系如下:
PCR回收产物 0.7 µg
green Buffer 5 μL
内切酶1 2.5 μL
内切酶2 2 .5 μL
ddH2O 补足50 μL
于37 ℃酶切约5 h或者过夜。
将酶切产物进行琼脂糖电泳并回收目的片段:
回收步骤如下:在紫外灯下,切下包含目的片段的胶条。用天平称量总重量并减去空管的重量算出凝胶的重量,按100mg=100μL来计算凝胶的体积,并加入3倍凝胶体积的QGbuffer置50℃水浴锅内将凝胶彻底融化。期间适当摇晃EP管,加快凝胶的溶解。等凝胶彻底融化后,加入与凝胶等体积的异丙醇并混合均匀。将上述液体全部转移到滤柱内,13000rpm离心30s。(可重复一次)然后弃掉管内液体,向柱内加入750μL的PE buffer。离心1min.。弃掉管内液体,再次空离2 min。换一个新的1.5 mL的EP管,向柱子内加入20 μl的ddH2O,离心1 min。为了提高回收率,可将溶解的DNA再次加入柱子内离心一分钟。弃掉柱子,即为回收的酶切产物片段,并测定浓度。
2. PHY-810载体的双酶切。
(1)将含载体PHY-810质粒的菌液过夜培养,并取新鲜菌液3-5mL提取质粒。具体方法参考QIAGEN质粒小抽说明书。
(2)取1µg新鲜质粒,用相应的限制性内切酶进行双酶切。酶切体系如下:
载体 1 µg
green Buffer 3 μL
内切酶1 1.5 μL
内切酶2 1.5 μL
ddH2O 补足30 μL
于37℃酶切约3h。
(3)将酶切产物进行琼脂糖凝胶电泳,电泳结束后,进行胶回收,方法同上。
3 过表达载体与目的片段的连接。
(1)测定回收载体和目的片段的浓度,目的基因扩增产物与PHY-810载体的体积比=(载体长度×0.02)/(目的片段长度×0.04)。
(2)过表达载体与目的片段的连接,连接体系如下:
回收载体 160 ng
目的片段 80 ng
5×CE Entry Buffer 4 μL
Exnase entry 2 μL
ddH2O 补足20 μL
于37 oC连接30 min后,立即置于5℃冰水浴冷却。
4. 转化。
(1)将感受态细胞置于冰上(4℃)待其自然解冻后,取10μL连接产物加 入感受态细胞中于冰上(4℃)放置30 min。
(2)之后于42℃水浴中热击90秒。然后迅速置于冰上(4 ℃)放置2-3 min。
(3)加入500μL不含抗生素的SOC培养基于37 ℃,225 rpm振荡培养45 min。
(4)3000rpm离心2 min,弃掉900 μL的上清液,将管底的菌液吹打散开, 加入到含有载体上对应抗性(氨苄或卡那等)的培养平板中,用灭菌的涂布器涂匀(涂布器的温度不能太高,以免烫死菌体),倒置于37℃恒温培养箱内过夜培养。涂板筛选重组质粒后挑取若干个单菌落,进行小量摇菌培养。
(5)筛选出质粒首先经酶切后电泳和PCR鉴定,进行菌液PCR初步鉴定,方法同上步骤3,只将模板替换成新鲜菌液2-3μl,鉴定证实PHY-PTH 构建正确(图3,图4)。
(6)将初步鉴定为阳性的制备的重组质粒DNA,选送测序公司进行测序鉴定。应用分析软件将测序后基因与文献发表的核苷酸序列进行同源性比较为95%,(图4)。
实施例二:表达验证
1. PHY-PTH转染 293T 细胞实验。
(1)转染前一天,接种适当数量的细胞至细胞培养板中,使转染时的细胞密度 能够达到 70~90%。
(2)对于每个转染样品,按如下步骤准备 Lipofectamine® 2000-DNA混和液。
a. 使用前,取10μL Lipofectamine® 2000转染试剂轻轻摇匀,用125μL
不含血清的优化,培养基(Opti- MEM)稀释,涡旋振荡2-3s充分混合试剂。
b. 用不含血清的Opti- MEM (125μL) 稀释 DNA(4µg)轻轻混和。
c.尽快将(a)稀释好的 Lipofectamine® 2000 与上述(b)稀释好的 DNA 轻轻混和,室培养5min,以形成 Lipofectamine® 2000-DNA 混和物。
(3)细胞在10cm培养皿,无血清培养基中进行转染,转染复合物添加24h后更换为含2%FBS血清培养基进行培养。
(4)将培养板置于37℃的CO2培养箱中孵育48小时后,west blot 检测细胞和细胞上清中FLAG标签的表达水平,免疫化学发光法检测细胞上清中甲状旁腺激素表达水平。
2. Western Blotting方法验证Flag表达。
(1)将玻璃板洗干净,固定于电泳槽上;配制 10%分离胶,加入 TEMED 后迅速灌胶,留出灌注浓缩胶所需空间(梳子的齿长再加 1cm),上部用水异丁醇封面。10%分离胶(15mL):
ddH2O 5.9mL
30%丙烯酰胺溶液 5.0mL
1.0mol/Ltris(pH6.8) 3.8mL
10%SDS 0.06mL
10%过硫酸铵 0.06mL
TEMED 0.006mL。
(2)配制 5%浓缩胶(6mL):
ddH2O 4.1mL
1.5mol/Ltris(pH8.8) 0.75mL
10%SDS 0.15mL
10%过硫酸铵 0.15mL
TEMED 0.006mL。
(3)灌注浓缩胶:待分离胶完全聚合后(约30 min),倾出覆盖层液,用 ddH2O洗涤凝胶顶部数次,以除去未聚合的丙烯酰胺,再用吸水纸吸净残留的液体,配制好浓缩胶后迅速灌入,并立即在浓缩胶液中插入干净的 Teflon 梳子,确保无气泡;待凝胶完全聚合后(30 min),小心取出梳子,清洗加样槽,将凝胶放入电泳槽内;将电泳缓冲液加入内外电脉槽中,使凝胶的上下端均能浸泡在缓冲液中,排除加样孔内可能的空泡。
(4)样品处理:将蛋白质样品与3×样品处理液,在一个子 Eppendorf 管中混合,95℃加热 10min,冰浴冷却,离心1 s,加样;加样:每孔上样的最大蛋白量:20~40μg。为了避免边缘效应,在未加样孔中加入等量的样品缓冲液。
(5)电泳:将电极插头与适当的电极相连,电流应流向阳极;将电压调至80V,样品的前沿迁移约 30-40 min。将电压改调至 120 V。待样品的前沿迁移至底部,关闭电源,拔掉电极插头,从电泳槽中取出凝胶玻璃板,小心移动两玻璃板间的隔片,将其插入两块玻璃板的一角,轻轻撬开玻璃板,凝胶贴于其中的一块凹槽上,切去浓缩胶部分和两侧没有加样的孔。
(6)蛋白质从 SDS 聚丙烯酰胺凝胶转移至固相支持体:PVDF 膜先泡于 100%甲醇,ddH2O 漂洗5min,右上角剪角。浸入电转移液中,注意驱除滤膜上的气泡;在一浅托盘中加入少量的电转移液,把滤纸浸泡于其中;将靠上方的电极(阴极)放于平层物上,石墨一边朝下,连接电源,根据凝胶面积按 0.65 mA/cm 2 接通电流,电转移 2 h;断开电源,拆除转移装置,去除各层,将 PVDF 膜浸泡于 TBST 中。
(7)封闭非特异性结合位点:5%脱脂奶粉 (TBST 配制),封闭。平放于摇床平台上,室温温育 1~2h。
(8)一抗: 取出膜在 TBST 中洗涤 1 次;将膜放入可加热封接的塑料袋中,加入适当浓度的一抗,4 ℃过夜。
(9)ECL 发光检测: 膜在 TBST 中洗涤每次 10min,共 3 次;将膜放入暗盒中,滴加 ECL 反取出应液(A 和 B 液等体积混合后立即使用),室温 1-3 min。在暗室用 X 线片曝光,显影、定影后观察结果。
(10)结果:重组体经脂质体法转染HEK293 48h 后, 经418筛选3周得到细胞抗性克隆,WEST BLOT 检测到FLAG在PTH重组质粒转染的293T细胞中成功表达(图5)。
3 全段甲状旁腺激素测定(免疫化学发光法)。
(1) 检测仪器
Atellicca IM 全自动化学发光免疫分析仪。
(2) 检测样本
PTH 质粒转染 293T 细胞上清。
(3)检测步骤
a 在反应杯中注入25μL样本;
b 再注入50μL标记试剂和100μL固相试剂,并在37℃下孵育8分钟;
c然后进行分离、抽吸,并用Atellicca IM清洗液清洗反应杯;
d 再注入酸性试剂和碱性试剂各300μL,以启动化学发光反应;
e 报告结果: 甲状旁腺激素结果单位为pg/ml(普通单位)或pmol/L(SI单位),换算公式为1pg/ml=0.106 pmol/L。全段甲状旁腺激素测定试剂盒的可测量的浓度范围为6.3-2000pg/ml(07-212.0 pmol)。
(4) 结果:重组质粒表达载体Pcdna3.1(+)-PTH转染的293T细胞,用化学免疫法在细胞上清中检测到PTH高表达(图6)。
SEQ ID NO. 1
人工合成的目的基因序列
ATGGGTGTGTGTATGTGCTGCTTTGAACCTATAGTTGAGATCCAGAGAATTGGGAGTGACATCATCTGTAACAATAAAAGAGCCTCTCTTGTGAAGATGATACCTGCAAAAGACATGGCTAAAGTTATGATTGTCATGTTGGCAATTTGTTTTCTTACAAAATCGGATGGGAAATCTGTTAAGAAGAGATCTGTGAGTGAAATACAGCTTATGCATAACCTGGGAAAACATCTGAACTCGATGGAGAGAGTAGAATGGCTGCGTAAGAAGCTGCAGGATGTGCACAATTTTGTTGCCCTTGGAGCTCCTCTAGCTCCCAGAGATGCTGGTTCCCAGAGGCCCCGAAAAAAGGAAGACAATGTCTTGGTTGAGAGCCATGAAAAAAGTCTTGGAGAGGCAGACAAAGCTGATGTGAATGTATTAACTAAAGCTAAATCCCAG
SEQ ID NO. 2
测序拼接结果如下:
agggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggatccgccaccATGGGTGTGTGTATGTGCTGCTTTGAACCTATAGTTGAGATCCAGAGAATTGGGAGTGACATCATCTGTAACAATAAAAGAGCCTCTCTTGTGAAGATGATACCTGCAAAAGACATGGCTAAAGTTATGATTGTCATGTTGGCAATTTGTTTTCTTACAAAATCGGATGGGAAATCTGTTAAGAAGAGATCTGTGAGTGAAATACAGCTTATGCATAACCTGGGAAAACATCTGAACTCGATGGAGAGAGTAGAATGGCTGCGTAAGAAGCTGCAGGATGTGCACAATTTTGTTGCCCTTGGAGCTCCTCTAGCTCCCAGAGATGCTGGTTCCCAGAGGCCCCGAAAAAAGGAAGACAATGTCTTGGTTGAGAGCCATGAAAAAAGTCTTGGAGAGGCAGACAAAGCTGATGTGAATGTATTAACTAAAGCTAAATCCCAGctcgaggactacaaagaccatgacggtgattataaagatcatgacatcgactacaaggatgacgatgacaagtagtgagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcc
备注:
小写碱基为载体序列,大写碱基为插入目的序列。
Claims (7)
1.一种人甲状旁腺激素真核表达载体重组质粒,其组成部件特征为:目的基因为人甲状旁腺激素(PTH1-84aa)基因(SEQ ID NO. 1);质粒载体为PHY-810,载体原件:CMV-MCS-3xFlag-SV40-Neo;双酶切位点为BamHI酶切位点和XhoI酶切位点,重组质粒载体大小为5888bp。
2.根据权利要求1所述的人甲状旁腺激素真核表达载体重组质粒,其构建方法特征在于包括如下步骤:(1)人工合成人甲状旁腺激素(PTH1-84aa)基因(SEQ ID NO. 1);(2)以所合成的PTH基因为模板,设计引物,P1含有BamHI酶切位点,P2含有XhoI酶切位点;(3)经PCR扩增PTH基因片段;(4)PCR扩增PTH基因片段用限制性内切酶BamHI和XhoI双酶切后,与经相同酶切的载体PHY-810连接;(4)将连接好的表达载体转化感受态细菌,涂板筛选、酶切测序鉴定,得到人PTH基因真核表达载体重组质粒(PHY-PTH)。
3.根据权利要求1所述的人甲状旁腺激素真核表达载体重组质粒,其结构特征在于人甲状旁腺激素基因克隆位点位于BamHI—XhoI之间,在不增加重组质粒载体bp数量的前提下,避开N端活性功能区,在C端连接flag-tag,使PTH与3xFlag融合表达,有利于提高重组蛋白质的产量、增强重组蛋白质的可溶性和稳定性,提高PTH功效和延长其作用时间。
4.根据权利要求2所述的人甲状旁腺激素真核表达载体重组质粒构建方法,其特征在于基于目的基因序列的引物设计,分别在上游引物的5’端插入一个BamHI酶切位点,在下游引物的5’端插入一个XhoI酶切位点:
p1(BamH I):CTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGGGTGTGTGTATGTG;
p2(Xho I):CATGGTCTTTGTAGTCCTCGAGCTGGGATTTAGCTTTAGTTAATACAT。
5.根据权利要求2所述的人甲状旁腺激素真核表达载体重组质粒构建方法,进行PCR扩增获得目的基因,其特征在于PCR扩增条件为:预变性94度,变性98度,2min,变性98度,10s,退火62度,30s,延伸68度,3min,30个循环,最后延伸68度,5min。
6.根据权利要求2所述的人甲状旁腺激素真核表达载体重组质粒构建方法,,其特征在于所述PTH片段与PHY-810载体的体积比=(载体长度x0.02)/(目的片段长度x0.04)。
7.根据权利要求1所述的人甲状旁腺激素真核表达载体重组质粒,其功效在于作为基因药物用于治疗甲状旁腺功能减退,本发明所提供的PHY-PTH作为基因治疗载体,可以使甲状旁腺外细胞表达PTH,从而起到替代甲状旁腺分泌PTH的治疗作用。
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