CN117305254A - 一种猪δ冠状病毒突变体病毒rPDCoV及其拯救方法和应用 - Google Patents
一种猪δ冠状病毒突变体病毒rPDCoV及其拯救方法和应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,涉及猪δ冠状病毒,特别是指一种猪δ冠状病毒突变体病毒rPDCoV及其拯救方法和应用。本发明利用基因工程方法,对PDCoV野生毒株基因组进行改造,改造的氨基酸位点为病毒S蛋白S173→P和Y179→C,将改造后的病毒基因组克隆到pGF载体上,通过单酶切,体外转录以及电转染LLC‑PK1细胞,成功拯救出猪δ冠状病毒突变体病毒rPDCoV。本发明将PDCoV HNZK‑02株生物基因组分为6个片段,基于酵母转化相关同源重组的方法一步得到感染性克隆,避免了传统反向遗传学技术中需要将病毒基因组各片段逐一连接,也避免了传统的连接方法中酶切连接效率低下的问题,大大提高了连接效率。
Description
技术领域
本发明属于生物技术领域,涉及猪δ冠状病毒,特别是指一种猪δ冠状病毒突变体病毒rPDCoV及其拯救方法和应用。
背景技术
猪δ冠状病毒(Porcine deltacoronavirus,PDCoV)是δ冠状病毒属中唯一能够感染哺乳动物的猪肠道冠状病毒,可引起10日龄以内的新生仔猪急性腹泻和死亡,病死率超过80%,是规模化养猪场中极易发生,且影响仔猪生长速率的主要病原体。另外,PDCoV具有跨种感染能力,可以感染7个种属的22种细胞系。2021年Nature报道,从海地三名发热儿童的血清样本中检测并分离出变异株hu-PDCoV,表明PDCoV具有感染人的风险。因此,如何有效防控PDCoV是我国畜牧业与社会发展亟待解决的重要问题。但是,目前仍缺少可用的商品化的疫苗来防控PDCoV感染,研制新一代疫苗仍然迫在眉睫。PDCoV的基因组RNA全长约25knt,基因组序列为5’UTR-ORF1a-ORF1b-S-E-M-NS6-NS7-N-3’UTR,分别编码两个大的多聚蛋白pp1a和pp1ab,刺突蛋白S、小膜蛋白E、膜蛋白M、辅助蛋白NS6、NS7、NS7a和核衣壳蛋白N。其中,S蛋白在病毒与细胞受体结合,在介导病毒入侵和感染中发挥重要作用,S蛋白某些氨基酸位点的突变,可能会使病毒在细胞中的生物学特征发生重大改变,从而为抗病毒疫苗的研发提供参考。
反向遗传学(reverse genetics)是通过构建病毒cDNA感染性克隆,实现在DNA水平对病毒基因组进行编辑,进而拯救重组病毒,探索病毒基因功能和生物学特性,被广泛用于病毒学致病机制、疫苗研发和药物筛选等研究领域。专利CN113308440A公开了通过制造N7-甲基转移酶缺陷型的冠状病毒,制备减毒疫苗毒株,该项目的设计思路是通过突变非结构蛋白的位点,实现甲基化酶的缺陷;专利CN114214338A公开了一种将猪德尔塔冠状病毒(PDCoV)的S基因替换EGFP基因,并成功拯救出能表达PDCoV S的重组PIV5病毒;然而并没有关于猪δ冠状病毒的拯救方法。
发明内容
本发明提出一种猪δ冠状病毒突变体病毒rPDCoV及其拯救方法和应用,本发明为PDCoV的病毒致病机制研究、疫苗开发提供了平台支撑,也为研究冠状病毒各种蛋白功能提供新思路。
本发明的技术方案是这样实现的:
本发明提出了一种猪δ冠状病毒突变体病毒rPDCoV以及拯救方法与应用,通过将PDCoV HNZK-02株的S蛋白第173位丝氨酸S和第179位酪氨酸Y分别突变为脯氨酸P和半胱氨酸C,然后将突变后的病毒基因组克隆到pGF载体上,该感染性克隆拯救出的猪δ冠状病毒突变体病毒rPDCoV感染LLC-PK1细胞后能引起细胞病变,且与PDCoV野生毒株相比,其在细胞上的复制能力增强,对仔猪的致病性减弱。该突变体病毒rPDCoV为猪δ冠状病毒新型弱毒疫苗的研发提供了新的工具。
具体来说:
本申请提供了一种猪δ冠状病毒突变体病毒rPDCoV,所述突变体与PDCoV野生毒株相比氨基酸突变位点为S173→P和Y179→C。
上述猪δ冠状病毒突变体病毒rPDCoV的基因组序列如SEQ ID No.19所示。
一种猪δ冠状病毒突变体病毒rPDCoV感染性克隆的构建方法,步骤如下:
(1)以猪δ冠状病毒的反转录产物cDNA为模板、序列表中SEQ ID No.1-SEQ IDNo.12所示序列为各片段扩增引物,扩增猪δ冠状病毒A-F片段,其中在A片段5’端首位插入T7启动子序列,F片段3’端末端插入ASCⅠ酶切位点序列,引物E-R和F-F包含突变体病毒rPDCoV S蛋白氨基酸S173→P和Y179→C突变位点,引物序列如序列表中SEQ ID No.10、SEQID No.11所示;
(2)以pGF载体为模板,SEQ ID No.13-SEQ ID No.16所示序列为引物,对pGF载体进行分段扩增,得到线性化pGF载体的G1和G2片段,pGF载体的核苷酸序列如SEQ ID No.20所示;
(3)在YEPD固体培养基上划线接种酿酒酵母VL6-48N,挑取单菌落到YEPD液体培养基中扩大培养,菌液中加入藤黄节杆菌酶(20T)和β-巯基乙醇裂解酵母细胞壁,制备酵母感受态细胞;
(4)将步骤(2)的G1片段和G2片段等比例混合,所得混合物与步骤(1)的A-F片段按1:1:1:1:1:1:1的质量比混合均匀,共转化至VL6-48N酵母感受态细胞中,将转化后的酵母细胞涂布于组氨酸(his)缺陷型固体培养基表面,待长出合适大小的乳白色菌落后,进行菌液PCR和核酸测序筛选出阳性cDNA感染性克隆,将最终确定的阳性克隆质粒用电转化法转化至EPI300大肠杆菌感受态细胞,进行大量扩增,获得足量的cDNA感染性克隆质粒,即猪δ冠状病毒cDNA感染性克隆。
利用上述猪δ冠状病毒突变体病毒rPDCoV cDNA感染性克隆的构建方法构建得到的猪δ冠状病毒突变体病毒rPDCoV cDNA感染性克隆。
上述猪δ冠状病毒突变体病毒rPDCoV 感染性cDNA克隆在拯救病毒中的应用。
进一步的,利用美国Bio-Rad公司生产的电转仪将权利要求8所述猪δ冠状病毒的全长mRNA和猪δ冠状病毒N蛋白的mRNA共转染LLC-PK1细胞,当出现典型病变时收取病毒上清液,获得拯救的突变体病毒rPDCoV。
上述的猪δ冠状病毒突变体病毒rPDCoV在制备预防猪δ冠状病毒新型疫苗中的应用。
本发明具有以下有益效果:
与PDCoV野生毒株相比,本发明提供了一种猪δ冠状病毒突变体病毒rPDCoV,具有以下优势:
(1)本发明利用基因工程方法,对PDCoV野生毒株基因组进行改造,改造的氨基酸位点为病毒S蛋白S173→P和Y179→C,将改造后的病毒基因组克隆到pGF载体上,通过单酶切,体外转录以及电转染LLC-PK1细胞,成功拯救出猪δ冠状病毒突变体病毒rPDCoV。对突变体病毒rPDCoV感染LLC-PK1细胞后进行TCID50测定;对感染突变体病毒rPDCoV的仔猪肠道进行病理切片检查。结果表明:拯救出的突变体病毒rPDCoV滴度为3.73*108TCID50/mL,感染仔猪肠道后,其致病性相较于PDCoV野毒感染组减弱,相比PDCoV野生毒株其生物学特性发生了改变。
(2)本发明提供的感染性cDNA克隆构建方法具有快捷、稳定、高效等优势。本发明将PDCoV HNZK-02株生物基因组分为6个片段,基于酵母转化相关同源重组的方法一步得到感染性克隆,避免了传统反向遗传学技术中需要将病毒基因组各片段逐一连接,也避免了传统的连接方法中酶切连接效率低下的问题,大大提高了连接效率。
(3)本发明提供的T7体外转录技术可获取足量的mRNA转录本,提高病毒拯救效率。本发明在病毒序列的5’端添加了T7启动子序列,在3’端添加了ASCⅠ酶切位点序列,通过酶切是感染性cDNA克隆线性化,采用体外转录的方式获得足量的mRNA,避免了直接转染真核表达质粒时,因细胞状态和转染效率低下导致产生较少量的mRNA,从而降低病毒的拯救效率。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为猪δ冠状病毒突变体病毒rPDCoV感染性cDNA克隆的构建示意图。
图2为猪δ冠状病毒突变体病毒rPDCoV基因组片段RT-PCR扩增的凝胶电泳图。
图3为猪δ冠状病毒突变体病毒rPDCoV和PDCoV野生毒株感染LLC-PK1细胞细胞病变示意图。
图4为猪δ冠状病毒突变体病毒rPDCoV间接免疫荧光鉴定。
图5为猪δ冠状病毒突变体病毒rPDCoV与亲本病毒的多步生长曲线。
图6为猪δ冠状病毒突变体病毒rPDCoV与亲本病毒感染仔猪空肠的IHC鉴定。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
必要的原料及试剂、培养基的来源或配制方法。
1、病毒、细胞、菌株及参考序列
PDCoV HNZK-02 (GenBank注册号:MH708123)分离保存于本实验室。LLC-PK1细胞、pGF载体、酵母菌株VL6-48N均由本实验室保存
2、主要试剂和仪器
ASCⅠ限制性内切酶购自New England Biolabs (Beijing) LTD;KOD OneTMPCRMaster Mix -Blue购自日本TOYOBO公司;RNA提取试剂盒、反转录试剂盒购自南京Vazyme公司;酵母质粒小量提取试剂盒、PCR纯化试剂盒购自Omega Bio-tek公司;酵母质粒大量提取试剂盒购自TIANGEN公司;mMESSAGE MMACHINE T7 KIT 25T体外转录试剂盒购自Thermo Fisher公司;PDCoV N蛋白单克隆抗体由本实验室保存;Goat Anti-MouseIgG H&L(FITC)、Goat Anti-Mouse IgG H&L (Alexa Fluor® 594)购自abcam公司;电转仪、PCR仪购自BIO-RAD公司;荧光显微镜购自Thermo Fisher公司。
实施例1:猪δ冠状病毒突变体病毒rPDCoV感染性cDNA克隆质粒的构建
构建策略如图1所示。根据猪δ冠状病毒HNZK-02株全长基因组序列(Genbank登录号:MT260150.1),利用Oligo 7.0软件设计6对特异性引物PDCoV-A-F、PDCoV-A-R、PDCoV-B-F、PDCoV-B-R、PDCoV-C-F、PDCoV-C-R、PDCoV-D-F、PDCoV-D-R、PDCoV-E-F、PDCoV-E-R、PDCoV-F-F和PDCoV-F-R,(SEQ ID No.1~12)RT-PCR分别扩增A、B、C、D、E和F六个片段,其中E片段下游引物E-R和F片段上游引物F-F包含突变体病毒rPDCoV S蛋白氨基酸S173→P和Y179→C突变位点。
表1、引物表
按照RNAfast200 总RNA极速提取试剂盒(上海飞捷生物技术有限公司)说明书提取PDCoV HNZK-02株的病毒基因组RNA。以病毒基因组RNA为模板,按照HiScript® II 1stStrand cDNA Synthesis Kit (南京诺唯赞生物科技股份有限公司)反转录试剂盒说明制备PDCoV HNZK-02株cDNA。反应体系为:模板RNA 7 μL,Oligo dT 23VN(50 μM)1 μL。反应程序为:65℃ 5 min,置于冰上骤冷2 min。之后反应体系为:上一步的混合液8 μL,2×RT Mix10 μL,HiScript Ⅱ Enzyme Mix 2 μL。反应程序为:50℃ 45 min,85℃ 5 min,4℃保存。
扩增A、B、C、D、E和F片段时,以PDCoV HNZK-02株cDNA为模板,分别用引物PDCoV-A-F/PDCoV-A-R、PDCoV-B-F/PDCoV-B-R、PDCoV-C-F/PDCoV-C-R、PDCoV-D-F/PDCoV-D-R、PDCoV-E-F/PDCoV-E-R、PDCoV-F-F/PDCoV-F-R进行扩增;反应体系为:cDNA 2 μL,上游引物(10 μM)和下游引物(10μM)各1.5 μL,KOD OneTMPCR Master Mix-Blue-(东洋纺生物科技有限公司)25 μL,ddH2O补齐至50 μL。扩增程序为:95℃ 3 min;98℃ 10 s,55℃ 5 s,68℃ 1min,循环35次;68℃ 5 min。目的条带大小分别为4352 bp,4625 bp,4461 bp,4789bp,1902bp,5620 bp,结果如图2所示。
以pGF空载(由本实验室提供,序列如SEQ ID No.20所示)为模板,分别用引物PGF-Vector-G1-F/PGF-Vector-G1-R、PGF-Vector-G2-F/PGF-Vector-G2-R进行PCR扩增,反应体系为:pGF cDNA 2 μL,上游引物(10μM)和下游引物(10μM)各1.5μL,KOD OneTMPCR MasterMix-Blue- 25 μL,ddH2O补齐至50 μL。扩增程序为:95℃ 3 min;98℃ 10 s,55℃ 5 s,68℃ 1 min,循环35次;68℃ 5 min。目的条带G1和G2大小分别为5468 bp和4239 bp。
将线性化的pGF载体的两个片段G1和G2与病毒A~F片段混合,通过化学转化法转化至酵母感受态细胞VL6-48N中。具体操作为:等比例将pGF载体的两个片段G1和G2与病毒A-F片段均匀混合;然后从-80℃冰箱中取出酵母感受态细胞200 μL的VL6-48N,在冰上融化,时间不可超过2 min;将上述混合物加入融化好的酵母感受态细胞VL6-48N中,用移液枪轻轻吹打混匀后室温静置10 min;加入800 μL PEG8000溶液并上下颠倒混匀,金属浴30℃孵育10 min;4℃ 590 g离心10 min,弃上清,加入800 μL 30℃预热的SOS液体培养基并轻轻吹打混匀,随后金属浴30℃孵育40 min;将上一步的混合培养液加入到熔化后的15 mL SORB-TOP-His固体选择培养基中,上下颠倒混匀后倒入到SORB-Bottom-His固体培养基平板上,待培养基凝固后,在30℃培养箱中培养3~7天至所有转化株可见。由于线性化的pGF载体的G1片段和片段A、片段A和片段B、片段B和片段C、片段C和片段D、片段D和片段E、片段E和片段F、片段F和线性化的pGF载体的G2片段、线性化的pGF载体的G1片段和线性化的pGF载体的G2片段之间分别存在有50 bp左右的末端同源臂,因此在酵母感受态细胞内,通过同源重组机制组装成携带PDCoV全长cDNA的感染性克隆重组质粒。PDCoV全长cDNA序列为:ACATGGGGACTAAAGATAAAAATTATAGCATTAGTCTATAATTTTATCTCCCTAGCTTCGCTAGTTCTCTACCGACACCAATCCAGGTGCGTCTGCCACCAAGTTGGCTACCCTTTCCAGGGGCGCTTCTGCGCTTGTTCACCATTAGATTACCTGGAAACCAGCCATTCAGGTTGGAGTTTCCCCAGGCTCTTTTGTGTGGGCATTAGCAGCTTGTGGTTTTTGCACAAAATCTAAGCTACTTACCGTTCCTCTGACCATCCACCACTTCTATAGACAGCACTGACTACCGTAGGGTTTAAGTCATACCGGTCTGCACCGCCCGTCAGCGGACACATTACCCAGCATAGCACTCCTTGCACCGAGCCTAGGTAGGATAAAACCCCCTACCGGGTGACTCTTAAGGCGTTTCCTCCACGGGATAGCCACTAGTCACTAGGTGTAAGTGATCTGATCTGGGCGTATTGTGTTGCGCAAGTGTGATACCCATAGGAGCGTGGAATCCTATTCTGCGGCTCAGTGCCTGATATAGCTGTGAAATGGCCAAGAACAAGTCCAAGCGCGACGCTATTGCGTTGCCTGAAAATGTACCACCACCTCTGCAACTTTTCATTCACGTTGCAGCTGCTGAAGAGGGTCACCCTAAGGTTACTACTTACCTTGGCAACTATAACCTCTATGCCACCAAGGCTCCGCCTGGCGTGCAGGTTCTTAGTGCTAAAACCTCTCTTACTGACTTTGAGAATGTCTTTGGAGCCCAACCCACCTTGCGATCAATTCGTAATCTGGTTTGTGAAGCTCGTTCGGCTGAATGGACAACTTCCAAGAATGCTTTTGCACTCAAAGCCACTCAACTTGACTACTCTGATGCCGTTTTGAGGGCAATGATTCGTTTCTGCCCTCCAAAGGTGTCCACACTCGCTGCCTTTGCTCTTTTTGGCCGATTGGTTAAAATTGAGGACAAGGAACTTGCTGAGTTAGCTCGTGATACTGCCCTTGAGTTGGCGTACACGGCTAAAATTGGTACATCTCTTGCTGACACGAGATCTGTCTCACTTATTCATAAGGACGCTTATCTAACTCTCAGTAATGAGGTTGTTGGCGTAACTTTTACTGCCGCACTTATGGCAAAGGCTACCACTGTTAATGGAGCAATGCAATACTCAAACTTTTACCTCTACCCTCGTGCCACTATTAAAGTGACTGATGGTAAGGCTGAAGCAATTGCAACTAAGCCTCTGCCTGCTGCCACTAAAGGCAAACCAATCACAGAGGATGTCAACCTTCTCCCTGACTATCAGCAGCTGCTTGTTGATCAAGTGACTGGCACTGAGGTTAAGGTTGGAGCTCTAACCTATGTTAAGACCACTGATTCACCACCCCTTTACTTTCCTAAAGTCAAGGGTGGTGTTATTGGTATTGCACTTAAGCAGCAGGGCACTGCGGCTAAGAAGCTCAATGTAGTCTTCCATGCTCAACCTGATGATGTTCTGCTAGCCTTCATACAACTTCAGCAATTCTTGAACCGTACTTCGGATTCAAGTGTTGAAATTACTGACTGCCAGAGTTATGAAGTATCTCCAACTGTGACGGTCAAAATTGGCCCGTCTAAACCTGGGGATGTCATCGTGGCTACTGATGAGGAATACCTTAAATGCTTTGAAACCCCTGAGGTAAGTAGGCTCTATAAGGTTTTCCAAACTCAATCTTGGGCTATCATTGAGCGTGCCTTCTCCAGTATGAAGATCCGCGTGTCCAAAGCTTTATCAGCATTTATAAGTTTTCTGCAAAACCTTGCAGATAACTTTACTGCAATAAGTGGTGTTGTCACTGCACTCATTCGTGAACTCCAGGATCTTACCCTGGATGTGGCGACACGTATCACTAACATACAATTTGTTTACCGTGCCGGTAAGCTTATTGTCGACACGACAAGTGTCATAGCTAAACTTTTCCAGCCATTTTGTGATTTTATATCACCTTTCCTTCGGAAAGTTGCTGGTTTTGCAATTTACACTGTTGGTAATCGCATGCTTATGTTTACCAGCACTGGCACCTTTCTTCTCACAAAGGCAACTACTAAGATACTCAATAAGGCAAAGTACATTTTTGATGTGGAGCCTGAGTACCCAGTAGATGTAACAACATCCAAAGTTGTAGTACATGAAGCACTCCAGCAAATCGACACTAAGCCCACTGGAGCTCTAGAGGCTGTTGATGTCGTTGTTGGTAATACTGTACTGCAAATGGCTACTGATGGCACTGCGTTCTACCCATCGGATGGTACGCACGCCTCTCTTCCAGGATTCAAAGCAGGCTCGGATGAGCTTTTCATAAGCTTCAACTGCGACCTCTTTGATGATGAGACTAATGCTCAAATCAACGAAACACTCGCTGCATATGAGCTTAACCAACTAGTGGCTCCAGGTGATTCTACACCGCGTCAAATTGCGACATTGGTTGTCGATACACTTGCAGATGCTATAACAGACCACTTTCCGGAGAAAACCATTGATCTACCTGAAGACTATCAAGTCTTTTCTGATCATGATGACCTCCCACTCGCACAATACCACATCCCTGATCACCTGAGCCTGTATATTCAGGCTATGGAAGGCGAAGATGATAGTGGTGATGAAATATGTATTGAGGACGATGATTACGACTGTCCTCAAGCCGACGAAGACACAGAAGGAGTAATTCCCCAACAGTGGGAACTTCCTGATGTTGATAAATTTTTACTCAAGATCCAGGAACGGAAGACCAGCAGCGACGAAGTACTTAGCGTCGACGTCTATCCTAAACCAGAGCCGGTCGGCAATGTTGGGATTGACGACAGCGCGTCGGAAAAGAAGCCAAATGGGGACCCAGTACCGGATCCTGAGGTCCATCCAACACTAGAGAGTGTGGATGTTGAACGACCAACCGAAACAGCAAACCAGGCTGTTGAAGACAAACCTTCTGATACCACCTTTGTGGTTGATGAGGAACAATTACAAGAATCAACACCAGAACATGAACTCCGCTCCTATGAAGGGGAGTTTGATTCTGATGATGAAATTATTATTCCTATAGTACCAGTAACACCTGCGGATTTAAAACCACAGACTATTACTATAAAGGAGTACTTTAAGTCTGAAAAACTTGAGACTATTAACGAAGGATCCACAGAGTCAGTTACACAATCTGACGATTCGTTTGACGAGTCATTTGTTGATGCTGAGTCTGATGATCCACAAGATCCTGCTGTATATGATGATACAACAATTATAACGGACAGCACTGATGTAGGCGATGAGCCTGAGACAACTCTAGCTACCATCGTTAACACACCTCTGACATTCGATAATAACTTGCCACCTGAAGCCATTAAACAACCCAGCCCGACTAAGGTTGAGTTAGTTGTTGGTGAATTGGCAAGTATTAAATTTGACAATTCTGTCTTAGTCAACCCTGCTAATGCGCAATTAACAAATGGCGGTGGAGCTGCCCGTGCAATTGCAAAATTAGCTGGTCCAAAATACCAAGAGTACTGTAATAGTGTGGCTCCTATCTCAGGACCGCTTACCACGGACTCTTTTGATGCTAAGAAACTTGGTGTAGCCTGCATCTTGCATGTGGTGCCACCCAAAGGTTCTGACCCTAATGTACAAGAACTCCTGTATCAAGCTTACAAGAGTATCCTTACTGAACCAGCACACTATGTTATACCTATACTAGGTGCTGGTATCTTTGGCTGCAACCCAGTCCACTCTCTGGATGCGTTCAGGAAAGCATGTCCAAGTGACATAGGTCGTGTCACCCTTGTCACTATGAACAAAAACCATTTGCAGGTGTGGGATGCTCTCAATAGGACCATTGTACGCACCACTACTGACTATGATCAAGTTACCACCAAGGCCCTTACACCCCAGGGAGTGTTAGAAGCCAATCTCTTTGATGGTGAGGACTTTGTTCAAGAACCAAAACCCGGTCAAATTTACCTTGAGGTTACTGAAGAAGTTCAGAACCAAGCCAAGGAACTTGACCTTAACCTTCAGCAATACTGCGTCTACCTGAAGACTTGCCACCATAAATGGGTTGTGAGTCGTACGAACGGGTTGATGCATTTAAAACAAAAAGATAACAATTGTTTTGTTAGTGCAGGTGTAAACCTGTTTCAAAACACTGCTTATCAACTTAGACCTGCTATTGATGCTCTCTATAGGGAGTATCTCAATGGTAATCCAAACAGATTTGTTGCTTGGATCTACGCATCCACTAACCGTCGTGTTGGTGAGATGGGTTGTCCACAGCAAGTTATTTCTTTGCTCGTTAGTAACTCTGACGCAGCATTTTCAGCAACTACAGCCTGTTGTAACACCTACTTTAACCACACAGGTGTTATTTCAGTAGCTCGTGAATATGACCCAATACAACCAAAGGTCTACTGCATGAAGTGTGATGTGTGGACTCCCTTTACACCCCAGAGTGGAAAAGGTGCAGTTGCAATTGGTACTTCTGCAGATGAACCTACAGGTCCTGCCATTAAATTTGCCGCAGCTCACTGCTGGTACACTAATGGCAAGAAAACAGTTAATGGCTATGACACTAAAGCTAATGTTGTAGCTACTTATCATAGGTTTGACGTGCCTAAGCCTCAACTTGTCGAGGACGTGGTTGCGCTGCCTACTAAAAATGACTTTGAAGTTCTCAATGTTGAAGAACTGCCACAGGATAGTGTGCTCCATTTGGACCCACCTCCTGTACAGGCCTTACAACCTAAGGCTAACCAACACATTGAGATTTTAGAAAACCCAGATTATCTGGACATTTTGGATCTTTGGATTCGTAAACCCAAATTCATCCTCGTAAAGTCGTGGAGTGTTTTGGGTAGAGCACTATGTAAGGCAGGTAAAGTTGTCTTTGTCAGTGCTTCGCTTTTGACCCGTTTCTACAATTACCTTGTAGAGATTGGTGCTCTTGACTCAACAATAAGGTTGTCAGTCGATCTTACCTGTAAATTTGTTAGAACGGTACTCCCATCGTCTAACACTGTACACAAAACTTGTCTTGGTCTGTATTATTCAGCCCAGACACTTTTTGTTTCTTTAGCACCATTCCTTATGTTACCAGCTGTAGTTAGTCTGCTTAATTCAGGCTATACAATTGGCACATATTTGTATGCAAAAACTGGCTGGCCTTGTAATTACAATGCCACGCAACACTTTGATTATAATTCTTACTGTGCAGGTGACTTGGTTTGTCAAGCCTGTTTTGACGGTCAAGACTCCCTACATTTGTATCCGCATTTACGTGTTAATCAGCAGCCCCTTCAGACCACTGACTACACTGTTTATGCGCTTTCACTAATACTACTATTAGCTAACATGACTCTTGTCATGGGTACGCTAATAGTTACTTTTTTTGTGAACTTCTATGGTGTGCAAATACCATTTTATGGTACACTTCTGATAGATTATCAGTCCGCGCTGATGATGACTTTCTCAGTGTACTACTTTTATAAGGTAATGAAGTTTTTCCGCCATCTCACACATGGATGTAAAATTCCAACGTGTATGGTATGTGCTAAACTTCGTACCCCACCTACTATAACAGTTGAGACTGTCGTTCAAGGCAGGAAATACCCATCTGTTATTGAAACAAATGGCGGGTTTACAATTTGTAAAGAACACAACTTCTATTGCAAAGACTGCTCTCTACAAACACCCGGCACTTTCATCCCGACAGAAGCTATTGAGTCGCTCTCACGAGCTACCAGGCTTAGTGTCAAACCAACAGCACCAGCATTCTTACTTGCTAGAGATGTTGAGTGCCAAACTGATGTTGTCGTTGCTCGCGCAATGCATAACCAAAATGCGCATGTGTGCATTTCAAAATACTCTGATATCCGTACCGTTGACCAACTACTTAAGCCTACTCCACTGTTTTCATACACTCCCGATGTTATCATCGCGGCAGACTTTGACAACAGAGGTAGTCTTAAGACAGCTAAAGAATTAGCTGTGGTTTTGTCAATGGACCTTAAACGTACTATAATTATCATTGATCAGGCCTATTCTAGACCCATTGATAATTATCAGGAAGTTGCTTCTCGTATTGAGAAGTATTACCCAGTTGCAAAGATTACACCCACAGGTGACATCTTTACAGACATTAAGCAAGCGACCAATGGCCAAGCTAGTGACTCTGCTATTAATGCAGCTGTTCTGGCTGTCCAGCGCGGTCTTGATTTTACAATTGACAACCCTAACAATATATTACCACATTACGCCTTTGACTTTTCAACCCTCAATGCAGAAGACCAGTCTACCATTTTGGAGAGTGGTTGTGCTAAAGGCAATCTCAAGGGCACTAATGTTGGTGTTGTTCTTTCAGCTAGCCTTGTTACACGTCTTAGTCAGCAGGCTATACGTGTGATTGCTAATGCTGCTTCACGTAATGGTGTAACATGCGCTGTTACTCCATCTACACTTGTTATGCGTGGGAATATTGCAACACAGCCCTTGACTCGCATCAAAGCTGGTGCACCTCCCATGCGTCAAAAAATTTTATGTGTTATCCTGGCACTTGCTATTGTGTACTTTGCTGCTATGGCTTTTGGCTTTTTGGCAAGTCAAATTACGCTTAATACAGTGCCTACGATTAAATCTGATATCCGCGCCTCTACCTTCTACGTTGTTAGAGATGGAGTCTTGGATACTGTTCGCTCAAATGACAAGTGCTTTGCAAATAAGTTTTTGGCATTTGATAGCTTCATTCAAGCACCTTACACTAATTCACCTGACTGTCCAGTCGTTGTGGGAGTTGTTGAAGTAACGACGCACTCTATTCCTGGAATTCCAGCAGGTGTTATTCATAGAGACGGTCTCATACTTAACATTTATGAACAGTCTCTTTATGAAACCCATCAGCGTCAGTCTATGGTTAGGGATGCGTTGTCACTTAAGACAGCAAATCTCTTTAACCTAGGCAAGCGTGTTGTAGTAGGATACACTCAACATGAAGTTGTTGTGGGTACCTCCTATTTTAATTCTCCTGCACTTTTTAACGCAAAGTGCACCTTCTTACAGTATCAGGACACTAGACAACTCTATTGCTATGATACTGTTCCTACTGAACATAAGCTTTACTCTGATGTGCTTCCGCACGTCGAGTATAAGGCTATTGACATTAATGGTGATCTTGTTCCTTTCAAGATACCGGAGCAGATAATGTTCTATCCACATATTGTGCGCTATACTAGCAATTCCTATTGCCGTATGGGGCATTGTTTTAATACTAACCCTGGTATTTGCATTTCATTTACGGACGAATTTCCGTATAGTGAAAATGTTAAACCCGGTGTATACTGTGCTGATACCTCTTTGCAGTTGTTTTCAAACCTCGTTTTGGGCACTGTATCTGGTATTCACATCTTTACATCAACAGCTGCATTGCTTGGATCTACTATTGTGATCATACTATGCGTTGTTGCTGTTCTTGCAGTTCAGCGATTCTTCAAGGAGTACACAACTTTTGTTATGTACACTTGTGGTCTTGCTCTTGTCAACATTGTGGGCATTGCACTTATGTACAAGTGCCTTGTCTTCGCGATCTTCTATTATGCAATCTACCTTTACTTTGTCCTTACTTTCCCCTCCTTTAAGAGGAATGTGGCATTGTTTTACTTCGCTGTAGTGATCGTGCCGCACGTGAGTAACATGCAATTGCTTGCGCTCATTGTGTGTAGCATTATCTACTTTCTCTACACCTATGTTCATACTGTAGCTAAGACAGCTGGGAAATTCTCTTCCTTCTTAGACGCAGCTAAAGCTACTTTTGTCATTGACAATGAAAAGTACGTGTTGCTTAAAGACCTCGCTGGTGCTGAATTTGACCAGTATCTGGCCTCTTACAACAAGTACAAATATTTTTCTGGTACTGCTTCTGATAAGGATTATGATAAGGTCTGTATGGCATTTCTTGCCAAGGCCTTGTCATCTTTTCGTGAAGGAGGCGGTTCACAGTTGTACACACCACCTAAATTTGCAGTTGTTCAGAGTCTTAAGACCAAGCTGCAAGCAGGTATCAAAATCCTCCTGCACCCTTCAGGTGTAGTTGAGCGATGTATGGTCTCAGTTGTCTACAATGGATCTGCATTGAATGGCATCTGGCTTAAGAATGTTGTCTACTGCCCACGCCATGTAATTGGAAAATTCCGTGGTGACCAGTGGACTCACATGGTCTCAATTGCTGATTGCCGCGACTTTATAGTCAAGTGTCCAATACAGGGTATTCAGCTAAATGTCCAATCAGTTAAGATGGTAGGAGCTCTCCTCCAGTTAACTGTTCATACCAACAACACAGCCACTCCAGACTATAAGTTTGAAAGGCTCCAACCAGGATCATCGATGACAATTGCTTGTGCTTATGATGGCATTGTACGACATGTCTATCACGTGGTCCTCCAACTTAATAATCTTATTTATGCAAGCTTCCTTAACGGAGCTTGCGGTAGTGTGGGTTACACTCTTAAGGGTAAAACACTCTACTTACATTACATGCACCACATTGAGTTTAACAACAAAACTCATAGTGGTACAGATCTTGAAGGTAACTTCTATGGCCCCTATGTGGATGAGGAAGTTATTCAGCAACAAACAGCATTCCAGTATTACACTGATAATGTAGTTGCTCAATTATATGCACACTTACTGACTGTTGATGCTAGACCAAAATGGCTGGCACAATCTCAGATAAGTATCGAGGATTTCAACTCATGGGCTGCTAACAATTCCTTTGCTAACTTCCCATGTGAACAAACTAATATGTCCTACATTATGGGACTCTCGCAAACTGCTCGAGTCCCCGTAGAACGTATCCTCAATACCATTATACAGCTAACCACCAATAGAGATGGTGCTTGTATTATGGGATCTTATGATTTCGAGTGTGATTGGACGCCAGAGATGGTATACAATCAGGCTCCAATTTCATTGCAGTCAGGAGTAGTTAAGAAAACTTGTACGTGGTTCTTCCACTTCTTGTTTATGGCTATTACCATGCTACTCGCTGCCATGCATGTCTTCCCTGTACACCTGTACCCAATAGTACTGCCATGCTTCACTGTCGTGGCATTCCTGTTGACTTTAACCATTAAACACACTGTTGTGTTTACCACTACATATTTGCTTCCGTCACTTTTGATGATGGTTGTAAATGCTAACACTTTTTGGATACCGAACACATTTCTGCGCACCTGCTACGAAACTATATTCGGTTCCCCAATTGCTCAGCGACTGTATGGTTACACTGTTGCTCTTTATATGCTGATCTATGCTGGACTTGCAATCAACTATACGTTGAAAACACTCCGGTATAGAGCAACTTCATTCTTATCTTTTTGCATGCAGTGGTTTCAATATGGTTATGTTGCACACATTGCGTACAAACTGCTTAATAAACCCTGGACAGAATCACTACTCTTCACAGCCTTCACAATGCTAACCAGTCATCCTTTGTTGGCTGCTCTTAGCTGGTGGCTAGCTGGTCGCGTAACTCTGCCCATTATCATGCCTGACTTAGCTATTCGTGTTTTGGCGTATAACGTCATTGGCTATGTCATATGTGTTCGATTTGGTCTTATGTGGCTTGCAAATCGATTCACAACTGTACCTATGGGCACATACCAGTATATGGTGTCTGTAGAGCAACTTAAGTACATGATGGCAGTTAAGATGTCCCCACCGCGTAATGCGTTTGAGGTGCTTATAGCCAACATTAGACTTCTTGGTTTGGGTGGAAACCGTAACATTGCTGTTTCTACTGTTCAAAACAAAATTCTTGATGCAAAAGCTACTGCTGTTGTTGTTGCTAACCTTCTTGAAAAGGCTGGCGTCACAAACAAGCACGCTATTTGCAAAAAGATTGTGAAACTCCACAATGATACCCTTAAAGCCACCACTTATGAGGAGGTTGAGGTAGCACTTGTGAAACTTCTTTCTCACATAATTGAGTTCTTGCCAACTGATCAGGTAGATGCTTATCTAGCTGATGCGGCCAATGCTCAACATGTTAATACCTATTTCGACAACTTGCTTGAGAACAAAGCTGTTGTTCAGGCTGTTGCCGATATCAACATTAATCTGGATTCTTATAGAATTTATAAGGAGGCAGATGCTATTTATAAACGATCTGTTGAGATGAACGAATCTCCACAGGAGCAAAAGAAAAAGCTTAAAGCTGTTAACATTGCAAAGGCGGAATGGGAGCGTGAGGCTGCTTCTCAGCGTAAGCTTGAAAAGCTTGCTGATGCTGCTATGAAGTCTATGTATCTTGCAGAACGTGCTGAGGATCGTCGCATTAAGCTAACCTCTGGACTTACTGCAATGCTTTACCATATGCTTAGACGTCTTGACTCAGATAGGGTAAAAGCTCTGTTTGAGTGCGCTAAGGCACAAATCTTGCCAATACATGCTGTAGTTGGAATTTCTAATGACAACCTTAAAGTTATTTTTAACGATAAGGATAGCTACTCTCATTATGTAGAGGGCAACACACTTATACATAAGGGAGTTCGCTACACTATTGTGAAGAAACTCTCCTTAGATAATGCACCTATTGAAGGCGTACCAGAAGAATTCCCTGTGGTCGTTGAGACTGTTAGGGAAGGTGTGCCCCAGTTGCAAAACAATGAGCTATGTTTGCGCAATGTTTTCACTGCTCAGAACACAGCTCAGGACTTCAATGGCAATGAATCCACTGTAAAATCTTTTTATGTTACTAGAACCGGTAAGAAGATTTTGGTTGCCATTACATCAACTAAAGACAATCTTAAGACTGTGACCTGTCTTACTGAGACCGGTAAGACAGTCCTTAACTTGGACCCTCCTATGCGCTTCGCACATACTGTAGGTGGAAAACAGTCTGTTGTCTATCTCTATTTTATTCAGAATATTAGTTCACTCAACAGAGGTATGATTATTGGCCACATCTCTGAAACTACTATCCTTCAGGCAAGTGGCACTCAAATTGAGTACCAGCAAAATGCCTCTCTTTTGACCTATTTGGCTTTCGCTGTAGACCCTAAGACAGCCTACCTTAAGCATCTTGCTGATGGTGGGTCTCCTATACAGGGTTGTATTCAGATGATTGCTACTATGGGTCCTGGATTTGCAGTTACTACTAAACCACAACCTAATGAGCATCAGTATTCTTATGGTGGTGCTTCAATTTGTCTTTATTGCCGTGCTCATATACCACATCCTGGTGTTGATGGACGGTGCCCCTACAAAGGCCGCTTTGTTCACATCGACAAAGATAAGGAACCTGTTTCCTTCGCCTTGACTCATGAGCCATGCAGTTCTTGTCAACGGTGGGTCAATTATGACTGCACCTGTGGATCTAGTCTGCAGAATTCGGCTTATTTAAACGAGTAACGGGTTCTAGTGACGCCCGGCTAGAACCCCTGCAGCCTGGAACTCAACCAGATGCTGTAAAAAGGGCCTTCCATGTGCATAATGATACCACCTCTGGTATATTCTTAAGCACAAAATCTAACTGCGCTCGGTTTAAAACCACACGCAGTGCCCTGCCTTTACCTAACAAGGGAGAGGTTGAATTGTACTTTGTTACTAAGCAGTGTGCAGCTAAAGTCTTCGAAATCGAGGAGGAATGCTACAACGCTCTTAGTACAGAGCTTTATACTACTGATGATACATTTGGTATCCTTGCCAAAACTGAGTTCTTCAAGTTTGACAAGATACCTAATGTCAATCGCCAGTATCTGACTAAATATACACTCCTGGACTTGGCTTATGCTCTACGTCATTTGTCAACATCTAAGGACGTTATTCAAGAAATTTTGATCACCATGTGCGGAACCCCTGAAGATTGGTTTGGGGAAAATTGGTTTGATCCAATTGAGAACCCATCCTTTTACAAGGAGTTCCATAAACTTGGGGATATTCTTAACCGTTGTGTTCTTAATGCCAATAAGTTTGCTAGTGCCTGTATAGACGCTGGTCTTGTTGGCATATTAACACCCGACAATCAAGACCTCCTGGGTCAGATCTATGACTTTGGAGATTTTATTATTACACAACCAGGTAATGGATGTGTGGACTTAGCATCCTATTATTCTTATTTAATGCCCATTATGTCCATGACTCACATGTTAAAGTGTGAGTGTATGGATAGTGATGGCAACCCACTTGAGTATGATGGATTTCAGTATGACTTCACGGACTTCAAGCTTGGCTTGTTCGAGAAATATTTTAAGTACTGGGACCGTCCTTACCATCCTAACACTGTTGAATGTCCAGATGACCGTTGCGTATTGCACTGTGCGAACTTCAATGTGTTGTTTGCTATGTGTATACCTAATACGGCATTTGGCAATCTTTGTTCAAGAGCTACTGTTGATGGCCACCTTGTGGTCCAGACAGTGGGTGTACACTTGAAAGAACTCGGTATAGTCCTTAACCAGGACGTTACCACACATATGGCAAATATTAATCTAAACACTCTATTGCGATTGGTAGGTGATCCCACCACCATTGCAAGTGTCTCAGACAAGTGTGTAGATTTAAGAACTCCTTGTCAGACCTTGGCTACTATGTCTAGCGGAATTGCTAAACAGTCAGTCAAGCCCGGGCATTTTAATCAACACTTCTACAAGCATTTGCTTGATAGTAACCTATTAGACCAACTTGGAATAGACATTCGCCACTTCTACTATATGCAGGATGGTGAAGCGGCTATCACAGACTACAGCTACTACAGGTATAATACCCCCACGATGGTAGATATCAAGATGTTCTTATTTTGCCTTGAGGTGGCAGATAAGTACCTTGAGCCCTACGAAGGTGGATGTATTAATGCACAGTCAGTTGTGGTTTCTAATTTGGACAAGTCAGCGGGCTACCCCTTTAACAAGCTAGGTAAGGCTCGTAACTATTACGACATGACTCATGCCGAGCAAAATCAACTGTTTGAGTATACAAAACGCAATGTTTTGCCTACACTCACTCAGATGAACCTTAAGTATGCAATTTCAGCCAAGGATCGTGCTCGCACTGTGGCAGGAGTGTCTATAATTAGCACCATGACTAACAGGCAGTACCATCAAAAGATGCTGAAATCTATTTCACTTGCACGCAATCAGACCATCGTGATTGGAACAACCAAATTCTATGGTGGTTGGGACAACATGTTACGACGACTGATGTGTAATATCAACAATCCCATTTTAGTGGGTTGGGATTACCCTAAGTGTGATCGTTCTATGCCAAACATGTTGCGCATTGCCGCTTCGTGCTTGCTAGCACGAAAACACACTTGCTGTAACCAAAGCCAGCGATTCTACCGTTTGGCTAATGAATGTTGCCAAGTACTATCAGAAGTGGTAGTCTCTGGTAACAACCTCTATGTAAAACCAGGTGGCACTAGCAGTGGTGATGCAACCACAGCTTATGCCAACTCGGTATTTAACATCTTACAGGTGGTTTCTGCTAATGTAGCCACCTTCTTATCAACTTCCACCACGACACATCTTAATAAGGACATTGCGGACTTGCATCGTAGTCTTTATGAAGATATTTATCGTGGTGACTCTAATGATATCACCGTCATCAATAGATTCTACCAGCATCTCCAAAGTTACTTTGGACTTATGATATTGTCTGATGATGGTGTCGCATGCATAGACTCAGCCGTTGCAAAGGCTGGAGCTGTTGCTGATCTTGATGGTTTCCGAGACATTTTGTTTTACCAAAACAATGTTTACATGGCAGACTCAAAGTGTTGGACAGAAACTGACATGAATGTTGGCCCTCATGAATTTTGCTCACAGCATACTGTGTTAGCAGAGCATGATGGTAAACCTTACTACTTACCTTACCCAGATGTCTCTCGCATTCTGGGTGCATGCATCTTTGTGGATGACGTTAACAAGGCTGACCCCGTTCAGAACCTCGAACGTTACATCTCACTTGCAATTGATGCATATCCCCTCACCAAGGTTGACCCTATTAAGGGTAAAGTCTTTTATTTGTTACTAGACTACATACGTGTCCTTGCTCAGGAGTTACAGGACGGTATCCTTGATGCTTTCCAATCACTCACTGACATGTCGTATGTAAATAACTTTATGAATGAGGCCTTTTATGCTCAGATGTATGAGCAAAGTCCTACACTACAGGCCAGCGGTGTTTGTGTGGTGTGTAATTCACCCACTATACTGCGCTGTGGTGATTGCATTCGTCGACCACTACTTTGTTGCGTCTGTGCCTACCAGCATGTTACGCAGACTACACATAAACGTATCATTGCTATCAACAACTACATTTGTAGTGTTGAGAATTGCAATGAGGACAATGTTGAAAAACTTTTCATTTCTGGCACTGCGATCTATTGTGAGAATCACAAACCCACGCTGTGCATACCCATTGTAGCTAATGGTTCTGTTTTTGGTATCTATCGCCACACTGCCCGTGGTAGTGATGACATAGACCTCTTTAACGAGCTTGCTACATCTAACTATGACACTATTGAACCTTATCAGAAGGCCAATCGTGCACCTTTATCACTTATGCTCTTCGCTGCTGAAACCATTAAGGCACTTGAGGAGTCTATCAAAAAGTCATATGCTACCGCAACCGTCAAGGATGTGTATGACCAACGCTTCATTAAACTTCTATGGGAACAGGGTAAAAAGCCGCCACCCATAACGAAGAACCACATTTTCACTGGCTACCATTTTAACAAGAATGGAAAAACCCAAGTTGGTGATTACATTCTTGCTAAAACAGATGGCAGTGACACTTATACTTACAGAGGAACATCTACCTACAAACTCCAAACAGGTGATGTTTTAGTCTTAATGGCACATGTTGTTACACCGCTCTCAGCACCCCCTGTGCTAACGCAGACAACATATGTCAGAAAATCACTTTTACCCGACTCTGTTGGTGCGTCTTATTATGTGCAACATTTTAAGTCATATAATGAGATAGCTATGCAGAGGGTTACAACAGTATTAGGTCCACCAGGCACAGGTAAGTCAACCTTTGCTATTGGTTTGGCTAAGTACTTTCCTAGTGCACGCATTTGCTACACTGCGTCTTCGCATGCAGCAATCGATGCACTCTGTGAAAAAGCTTTCAAGACAATACCTGTAGGCCAATGCAGTCGTATCGTACCCACACGTACAACTGTTGAGTGCTTTCAGGAGTTTGTCGTAAATAACACAACTGCACAGTATATCTTCTCGACTATCAATGCCTTACCTGACATTAAGTGTGACATTGTAGTTGTAGATGAGGTTTCTATGTTGACCAATTATGAGCTTTCCTCTGTGAATGCTCGTTTGGTTTACAATCACATTGTGTATGTTGGTGATCCTTATCAGTTACCTTCACCTAGAACTATGCTTACGTCTGGCCAGCTTTCGCCAGCTGACTATAACGTAGTTACTGATATAATGGTACATGCAGGAGCGGATGTTATGCTCGACATGTGCTACAGATGCCCACGTGAAATCGTTGAGACAGTGTCTAAACTTGTCTACGATAACAAACTAAAAGCGGCGAAACCGAACTCAAGACAGTGTTACAAGACCATTGTGAACTTTGGTCCTGGAGACGTTGCTCATGAGGGACAATCTGCCTACAACGAAGCACAGTTGCGTTTCGCACTCGCATTTAGACAACAAAAGCGGTGGGATAACGTGACTTTTATATCTCCATATAATGCTATGAATGTGAAAGCATCCTTAGCAGGTTTCTCTACTCAGACCGTTGACTCTTCTCAAGGTTCTGAGTATGATTATGTTATCTTTTGCGTGACCACTGACTCAGCACACGCACTTAACATGGCTCGTTTAAACGTTGCCCTTACACGCGCAAAGATAGGTATCCTTGTGGTGTTTAGGCAGGCAAACGAACTTTACAATAGTTTGCAGTTTGAATCTATTGATTCACAGCTTCAGTCGAGTGCTGAGAGAAACCTCACACCACTGTTTAAGCGCTGCGGCTATGAGTATAATGGCGTCCATCCAGCTCATGCTTTGACCTGGCATGATTGTGGTGCAGAGTACCGCTGTGAGGAGCCACTTGCTAAATTAGTAGGAGTTGCCGATGGCACTCTTATATCATACAAAACCCTAGTATCCACACTTGGGTTTCTTCCATCACTTAAAATTGATGCATATCATAATATGTTCCTAACACGTGACGCGTGTCGCACCTATGTTCAGAGTTGGATCGGCATAGACGTTGAAGCAGCACACGCCATAAAACCTAACACCGGGACTAACCTGCCATTGCAAATAGGTTTTAGTACCGGAAAGAATTTTTCAGTTACTCCAGAGGGAATTTGGGTAAACGAGCACGGATCTTGCACTGAGCCCGTCCCTGCCAAAATACCTCCTGGAGAACAATTTCGTCACCTTAAGAAGGACATGCGCCAGGCGCGTCCTTGGAAGGTTGTTCGACGTGAGATTGCTACTCACATTGCTGAGGTAGCTCCTCACACTGATTATATATGCTTTGTCACTTGGGCTCACCAGCTTGAGCTAGCGACAATGCGCTACTTTGTCAAACTAGGTATGGAAGAAAAATGCTTTTGTGGCAGGCGGGCTTGTTTCACTAATGGAACTGAGTTCGCTTGCAAAGCACACCATTCTCTCACCATTCCACAATGTGATTATGTGTACAATCCATTCCTCATCGACGTGGCTACGTGGGGATTCTCGGGACGGCTTTCCACCAACCATGACGCGGTATGCACATATCATGCTAATGCCCATGTTGCATCAGCTGATGCAATCATGACGGTATGTTTAGCTATCCATGAACTGTTCAGTACTGTTGACTGGAACCTTGAATTTCCAGTAACTGCTGAGCAATCGCAACTTAACAAGGCCTGTCGCTTAGTACAGGCAAACTACTTAAATATACTACTCACTACAACCAAAGCCACGGTGGTTCACGATATTGGTAACCCAAAAGGTATCCCTATCGTGCGCAAACCTGGTGTTAAATATCACTTCTATGATCAAGCACCCATTGTCAAACACGTTCAAAAACTAAAGTACAAGCCAGAGATGGAGGCCCGTTTCACCGATGGTTTGACTATGTTTTGGAATTGTAATGTTGACACATACCCTGCTAACGCCCTTGTGTGCCGCTACGACACTCATCGGCAGAAGCATTTAATTGGACCTAATGGTTCAGCACTATATGTTAATAAGCATGCTTTTCTCACCCCTGAGATGCATACTTATGCCACACATAAACTCAACTTGGCTCCACTCATCTACTACTCCACCACAGATTGTAGTAGTGAACAGCCTATTGTTGTTACTTACAGAGATTGTGTCACCCGGTGCAATACTGGAAAAACTCTCTGTCCAAATCATGCTCTTGAATACCAAGAGTTTATCAATGCATACAATCTCATGGCTCGCCATGGATTTAATGTTTACATACCACGCAATGTCAACATCTACAACTGTTGGCTTACTTTCACTAATCTCCAAAACCTTGAAAACTTAGCTTACAACTGTTATTATAAGAACTGCAATGCTCACGTTGATGGGCAGCTTGATGTAGTTATTAATAATAACGCTGTATATGCTAAGGTCGACAATAATCTTGTCAAACTTTTCGACAACCGCACTAATTTACCTGTCTCAGTGGCTTTTGAACATTACACTAACAGGCATACCCGTTCACTGCCAACTACACAGCTGTTATCTGGTTTAGGCGTAACCGCCACCAGAAATTTCACTGTGTGGTTTGACAATGATACAATTTTCCAATACACTATTAATGTATCTACGTATACTGACATCGACCCTAGTACCCATGTTGTCCTCTGTGATGATAGGTACGGAACAGATTGGAGTCAGTTTAACCAACTTCCTAATGCAGTATTCCTCACCAAAACTAAGGTGAAGAAAACAGAACCGTTTGTTTGTACAGCACTGACCCTAAATGGCCTCGCCATTGACGGTGAAGAGCTATACATCTATGTACGCTATAACAATCAACTGACCACATTTGCTACTACTTGTACACAGGGTAGAAATGTTGAGCAGTTTATACCTAAAACACCTATGGAAAGAGACTTCCTCGAGATGTCTCAACAGTCCTTCATCCAGCAATATCAATTGCAGGAACTGGGTGTTGAACACATTATTTATGGTGATGATTCCAGTCCAGTCATTGGCGGAACTCACACACTTATTTCACTAGTTAAAAACAAGTTTGAACATCAGCTTGTCAACCATGTTTACAACCCAGTCCAGAACTGTGTTGTTACCTCACCTAACGCAAGCTCCAAGAACGTTTGCACTGTTCTTGATGTTCTTCTTGATGACTACATTGACATCATAAGACAAGCACATGCCAGTTACACAAGTAAATCCAAAGTATTCACTGTGTCAATTGACAACCAACAAATTAGATTCATGCTTTGGCATGATGAGCAAGTCAAGACTTGCTACCCAATCTTACAGTCACTTACCAATGGTTACCAGATGCCATCTGTGTACAAAACATTGGTTACTGACTTACAACCAGCTGACATCCCTAATTATCATTCCTACACCCCTCGGGTGCCTGGAGTAGTTAAGAATGTTATCAAGTACCGACAACTTTTCAACTACATAGTTAAAAAGGATAGGTTGGCAGTACCACACAATATGACCGTATTACACCTTGGAGCTGCATCTGCACTAGGTACAGCACCAGGTTCTTCAGTCATAAAACAAATGTTTCCTGAAGGAACTGTTCTTATTGATCTTGATATAAGAGAGTTCACTTCAGATGCTAACCAAATAATAGTTACAGACTACAGAACTTACATACCACCACACCACGTAGACGTCATATTTTCTGACCTCTACTGTTGTGATGACATACACTTCTTTGACAATCTAATAAGGATAGTTAAGGAGAGGCTCGCCCTCGGTGGTTCTATCTTTGTTAAGATAACTGAACATTCATTCTCACCTGAACTCTACTCACTTGCGGGTTGGTTCGATGATTATCAACTATTTTGCACAGCAGTTAATGCCTCGTCTTCAGAAGCATTTCTATGCTGTTTTAATTATTTGGGGCATGCTAAGGAAAACATTAATGGTTTTAACTTACATGCTTCCTACATTCAATGGCGCAATGAAATAGCGTTGACACCAACCTATTCTCCTTTAGCGGACAACCCGGCTACGGCCTGTAAGCTAAAAGCAACGCCTATTATCTCGGCTCGTGAGTTAGAGAAGAAGCCTATTCTTCGCTATCTCGTTGCATCAGGACGCCTTCTTGTGAGGCCACCAGAATGCAGAGAGCTCTATTGATTATGACCTTACTTTGTCTCGTTCGAGCAAAGTTTGCTGATGATCTACTCGATTTGCTCACCTTCCCGGGTGCACATCGCTTCTTACATAAACTCACGAGTAATTCCAGCAGTCTCTACTCGCGGGCTAATAACTTTGATGTTGGCGTTCTTCCTGGCTACCCCACTAAGAACGTTAACCTCTTCTCACCACTTACTAACTCTACTTTGCCAATTAATGGCCTTCATCGGAGTTACCAACCACTCATGCTGAATTGTCTTACTAAAATAACTAACCACACTCTCAGCATGTATCTCCTACCTAGTGAGATACAAACTTATAGCTGCGGCGGTGCCATGGTTAAATACCAGACACATGATGCAGTTCGTATCATTTTAGACCTCACTGTCACTCACCACATCTCTGTTGAAGTCGTTGGCCAACATGGTGAAAATTATGTGTTTGTTTGTAGTGAGCAGTTTAACTATACCACTGCATTACACAACTCTACCTTCTTCCCACTTAATTCTGAGCTTTGTTGCTTTACTAATAACACCTACTTAGGTATTCTTCCACCTGATTTAACTGACTTTACGGTCTATCGTACTGGGCAGTTTTATGCTAATGGTTACCTTTTAGGTACTTTACCTATTACGGTTAACTATGTAAGGTTGTATCGGGGTCATTTGTCGGCCAATAGTGCCCACTTTGCCCTTGCAAACCTAACCGATACACTCATAACACTTACCAATACTACTATATCGCAAATCACTTATTGTGATAAGTCAGTAGTTGATTCAATAGCATGCCAGCGCTCTTCTCACGAAGTGGAGGATGGGTTTTACTCTGACCCTAAATCTGCCGTTAGAGCTAGGCAACGTACTATTGTTACACTACCTAAGCTCCCTGAGCTTGAAGTAGTGCAGCTAAATATTTCTGCACACATGGATTTTGGCGAAGCCAGACTTGACAGCGTTACCATCAATGGTAACACATCCTATTGTGTCACTAAGCCTTACTTTAGGCTTGAAACTAACTTTATGTGTACAGGTTGCACTATGAATCTGCGCACTGATACCTGTAGTTTTGACCTGTCAGCAGTAAACAATGGCATGTCATTCTCTCAATTTTGTCTAAGCACTGAATCTGGTGCTTGTGAGATGAAAATTATTGTTACCTACGTATGGAATTACTTGCTAAGGCAGCGTTTGTATGTTACTGCTGTAGAGGGCCAGACTCACACTGGAACCACTTCAGTACATGCAACAGACACTTCTAGTGTAATCACTGATGTCTGCACTGACTACACTATCTATGGAGTCTCTGGTACTGGCATTATTAAGCCATCAGATCTCTTATTGCACAATGGCATAGCATTCACCTCTCCAACAGGTGAGCTTTATGCATTTAAAAATATAACCACTGGCAAAACCCTTCAAGTCTTACCGTGTGAAACCCCTTCTCAACTGATTGTGATAAATAACACCGTTGTTGGTGCTATCACATCCAGTAACTCAACTGAAAATAATAGGTTTACTACTACTATTGTCACACCTACTTTCTTTTATTCCACAAATGCCACCACCTTCAACTGCACCAAGCCTGTTTTGTCCTATGGACCCATCAGCGTGTGTAGTGATGGTGCAATTGTGGGAACATCCACATTACAGAATACTCGACCATCCATAGTTTCACTATACGATGGCGAAGTTGAAATACCATCTGCATTTTCCCTTTCTGTTCAGACGGAGTATTTGCAAGTTCAAGCAGAGCAAGTTATAGTTGATTGTCCTCAGTATGTATGCAATGGCAATAGCCGTTGTCTACAATTACTGGCACAATACACCTCAGCTTGCTCTAACATTGAAGCAGCTCTGCATTCCTCTGCACAGTTGGATAGCAGAGAGATTATAAATATGTTTAAAACATCAACACAGTCCTTGCAGTTAGCTAATATTACCAACTTCAAGGGTGACTACAATTTTAGCAGCATACTAACCACCAGACTTGGTGGCAGATCTGCTATTGAAGACCTTCTTTTTAATAAAGTTGTTACTAGTGGCCTTGGCACTGTTGATCAGGACTACAAAGCCTGCTCTAGAGACATGGCCATCGCTGACTTAGTTTGTTCCCAGTATTACAATGGCATCATGGTTCTACCTGGTGTTGTTGATGCTGAGAAAATGGCAATGTATACTGGCTCTCTTACTGGAGCTATGGTATTTGGAGGACTGACTGCCGCAGCGGCAATACCATTTGCTACGGCAGTACAAGCCCGCCTCAATTATGTCGCACTGCAAACAAATGTACTACAAGAAAACCAGAAAATTCTTGCAGAATCATTTAACCAAGCAGTTGGCAATATATCACTTGCACTATCTTCTGTTAATGATGCCATCCAGCAAACTTCTGAGGCTCTTAACACCGTAGCTATTGCTATTAAAAAGATTCAAACAGTTGTTAACCAGCAGGGTGAGGCGTTATCACACCTGACTGCACAGCTGTCAAACAATTTTCAAGCAATTTCGACTTCTATTCAAGACATTTACAATCGTCTTGAGGAAGTAGAGGCTAACCAGCAAGTTGACCGTCTCATCACAGGACGGTTGGCCGCACTTAATGCATATGTTACTCAGTTACTCAATCAGAAGTCTCAGATTAGACAATCTCGATTGTTAGCTCAGCAAAAGATTAATGAGTGTGTCAAATCTCAGTCGTCCAGATACGGTTTCTGTGGAAATGGCACACACATCTTCTCACTTACACAGACTGCACCAAATGGCATATTTTTCATGCATGCAGTGCTAGTACCCAACAAATTCACACGTGTCAACGCTTCTGCCGGCATTTGTGTGGATAATACGAGAGGCTACTCATTGCAGCCTCAACTTATACTCTACCAGTTTAATAACTCCTGGAGAGTTACACCTAGAAATATGTATGAACCCAGACTGCCCCGGCAGGCTGATTTCATACAATTAACTGATTGCAGCGTTACTTTTTACAACACCACTGCTGCTAATCTTCCCAATATTATCCCTGACATTATAGATGTTAATCAAACAGTCAGTGATATTATTGACAATTTACCTACAGCAACACCTCCTCAGTGGGATGTTGGTATCTATAACAACACTATTCTCAACCTCACCGTTGAGATTAATGATCTACAAGAGCGGTCTAAAAACCTCTCACAGATTGCAGATCGTTTACAAAATTATATTGACAATCTTAACAATACTCTAGTTGACCTTGAATGGCTCAACAGAGTAGAAACTTACCTTAAATGGCCGTGGTATATATGGCTTGCCATTGCCCTGGCTCTTATTGCATTTGTGACAATCCTCATAACAATCTTTCTTTGTACTGGTTGTTGTGGTGGTTGCTTTGGTTGTTGTGGCGGTTGTTTTGGCCTTTTCTCTAAGAAGAAAAGGTATACCGACGACCAACCAACACCGTCCTTTAAGTTTAAGGAATGGTAGTCGACGACTGGGCCGTTACCATCCCTGGACAATATATTATTGCTATACTAGTTGTCATCTGCATTGGTGTGGCACTACTTTTTATTAACACTTGCTTAGCTTGTGTTAAATTATTTTACAAGTGCTACCTAGGGGCAGCATACCTTGTTAGGCCTATTATAGTGTACTACTCCAAGCCGAACCCCGTACCTGAGGATGAGTTTGTAAAAGTACACCAATTTCCTAGAAACACTCACTATGTCTGACGCAGAAGAGTGGCAAATTATTGTTTTCATTGCGATCATATGGGCGCTTGGCGTCATCCTCCAAGGAGGCTATGCCACGCGTAATCGTGTGATCTATGTTATTAAACTTATTCTGCTTTGGCTGCTCCAACCCTTCACCCTAGTGGTGACCATCTGGACCGCAGTTGACAGATCATCTAAGAAGGACGCAGTTTTCATTGTGTCCATAATTTTTGCCGTACTAACCTTCATATCCTGGGCCAAGTACTGGTATGACTCAATTCGCTTATTAATGAAAACCAGATCTGCATGGGCACTCTCACCTGAGAGTAGACTCCTTGCAGGGATTATGGATCCAATGGGTACATGGAGGTGCATTCCCATCGACCACATGGCTCCAATTCTCACACCAGTCGTTAAGCATGGCAAGCTCAAGCTACATGGGCAAGAGCTGGCCAATGGCATATCAGTTAGAAATCCGCCACAGGATATGGTGATAGTGTCACCAAGTGACACCTTTCACTACACTTTTAAGAAACCTGTGGAATCAAACAACGATCCAGAATTCGCTGTTCTGATATACCAGGGTGACCGCGCTTCAAACGCTGGACTTCACACCATAACCACTTCAAAGGCCGGTGACGCTCGCCTGTATAAGTATATGTAATGTGCAACTGCCATCTGCAGCTGCGAGATTTATATAGATTGTGCAATAAGCTGCACATCAGAAGAGACGATGTTCCTGAGCTTATTGACCCTCTCGTTAAAACTCGTTGTTTTGCTTACAGTCTCGTGGTTCTTGCTAATGCTAATCCAATTGCATTTAGCATACTACCTCGGAAAATTCTTATCAATGGTGAGCCTTTACTGCTTGAATATGGTAGCATATATGGTAAAGACTTTATCATTCGACCATCGCTCCAAGTCATTCTTGAAGATGAATTAAATTAAAGTTTTGACACCAATCTATCATGGCTGCACCAGTAGTCCCTACTACTGACGCGTCTTGGTTTCAGGTGCTCAAAGCTCAAAATAAAAAGGCTACTCATCCTCAGTTTCGTGGCAATGGAGTTCCGCTTAACTCCGCCATCAAACCCGTTGAAAACCATGGCTACTGGCTGCGTTACACCAGACAAAAGCCAGGTGGTACTCCGATTCCTCCATCCTATGCCTTTTATTATACTGGCACAGGTCCCAGAGGAAATCTTAAGTATGGTGAACTCCCTCCTAATGACACCCCAGCAACCACTCGTGTTACTTGGGTTAAGGGTTCGGGAGCTGACACTTCTATTAAGCCTCATGTTGCCAAACGCAACCCCAACAATCCTAAACATCAGCTGCTACCTCTCCGATTCCCAACCGGAGATGGCCCAGCTCAAGGTTTCAGAGTTGACCCCTTCAACGCTAGAGGAAGACCTCAGGAGCGTGGAAGTGGCCCAAGATCTCAATCTGTCAACTCCAGAGGCACAGGCAATCAGCCCAGGAAACGCGACCAATCTGCACCAGCTGCGGTACGTCGTAAGACCCAGCATCAAGCTCCCAAGCGGACTTTACCCAAGGGTAAAACCATTTCTCAGGTATTTGGCAACCGGTCTCGCACTGGTGCCAATGTCGGCTCTGCAGACACTGAGAAGACGGGTATGGCTGATCCTCGCATCATGGCTCTAGCCAGACATGTGCCTGGTGTTCAGGAAATGCTTTTCGCTGGCCACCTTGAGAGCAACTTTCAGGCAGGGGCAATTACCCTTACCTTCTCTTACTCAATCACAGTCAAGGAGGGTTCTCCTGACTATGAGAGACTTAAGGATGCGCTCAATACGGTCGTTAACCAGACCTATGAGCCACCCACCAAACCAACTAAGGACAAGAAGCCTGACAAACAAGACCTGTCTGCTAAACCCAAACAGCAGAAGAAACCTAAAAAGGTAACTCTGCCAGCAGACAAACAGGATTGGGAGTGGGATGATGCTTTTGAGATAAAGCAGGAATCAGCAGCGTAGACATCACTCTATGTCTGTTAAACCCACCCAATTCCACTCAAATATCTCTTTGGTTTCAGAGAGTCATAGTGTATAGCCAGAGAGCCAGTCAGAGGGCGCTATCATGCAAACTAGGGCTGGCTACTCTAGCACAGAATCACATCCCGATAATCAACAGTGCTAGAAGGTTGATTATACCATTTAATATGCCGAGGCCACGCGGAGTACGATCGAGGGTACAGCATAATCTCAACTTTTGTTGAGCCACAATTTTAATCCTAATTGGAGAAGGCCAAAGGACTGTACTACTTTTGTAGGTGTAGCAGTCGCCCAGTGGGAAAGCGCCAACTAGGTTACAATTGTGGTGGGGACAAATTAGGGGAAATTAAATTGGCTTATAGGGGGGATGGAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
pGF载体的序列为:GAGTCGACCTGCAGGCATGCAAGCTTGAGTATTCTATAGTCTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGAACCCCTTGCGGCCGCCCGGGCCGTCGACCAATTCTCATGTTTGACAGCTTATCATCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGCGATAAGCTCATGGAGCGGCGTAACCGTCGCACAGGAAGGACAGAGAAAGCGCGGATCTGGGAAGTGACGGACAGAACGGTCAGGACCTGGATTGGGGAGGCGGTTGCCGCCGCTGCTGCTGACGGTGTGACGTTCTCTGTTCCGGTCACACCACATACGTTCCGCCATTCCTATGCGATGCACATGCTGTATGCCGGTATACCGCTGAAAGTTCTGCAAAGCCTGATGGGACATAAGTCCATCAGTTCAACGGAAGTCTACACGAAGGTTTTTGCGCTGGATGTGGCTGCCCGGCACCGGGTGCAGTTTGCGATGCCGGAGTCTGATGCGGTTGCGATGCTGAAACAATTATCCTGAGAATAAATGCCTTGGCCTTTATATGGAAATGTGGAACTGAGTGGATATGCTGTTTTTGTCTGTTAAACAGAGAAGCTGGCTGTTATCCACTGAGAAGCGAACGAAACAGTCGGGAAAATCTCCCATTATCGTAGAGATCCGCATTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATTTGAATATGCCTTCAGGAACAATAGAAATCTTCGTGCGGTGTTACGTTGAAGTGGAGCGGATTATGTCAGCAATGGACAGAACAACCTAATGAACACAGAACCATGATGTGGTCTGTCCTTTTACAGCCAGTAGTGCTCGCCGCAGTCGAGCGACAGGGCGAAGCCCTCGAGCTGGTTGCCCTCGCCGCTGGGCTGGCGGCCGTCTATGGCCCTGCAAACGCGCCAGAAACGCCGTCGAAGCCGTGTGCGAGACACCGCGGCCGGCCGCCGGCGTTGTGGATACCTCGCGGAAAACTTGGCCCTCACTGACAGATGAGGGGCGGACGTTGACACTTGAGGGGCCGACTCACCCGGCGCGGCGTTGACAGATGAGGGGCAGGCTCGATTTCGGCCGGCGACGTGGAGCTGGCCAGCCTCGCAAATCGGCGAAAACGCCTGATTTTACGCGAGTTTCCCACAGATGATGTGGACAAGCCTGGGGATAAGTGCCCTGCGGTATTGACACTTGAGGGGCGCGACTACTGACAGATGAGGGGCGCGATCCTTGACACTTGAGGGGCAGAGTGCTGACAGATGAGGGGCGCACCTATTGACATTTGAGGGGCTGTCCACAGGCAGAAAATCCAGCATTTGCAAGGGTTTCCGCCCGTTTTTCGGCCACCGCTAACCTGTCTTTTAACCTGCTTTTAAACCAATATTTATAAACCTTGTTTTTAACCAGGGCTGCGCCCTGTGCGCGTGACCGCGCACGCCGAAGGGGGGTGCCCCCCCTTCTCGAACCCTCCCGGTCGAGTGAGCGAGGAAGCACCAGGGAACAGCACTTATATATTCTGCTTACACACGATGCCTGAAAAAACTTCCCTTGGGGTTATCCACTTATCCACGGGGATATTTTTATAATTATTTTTTTTATAGTTTTTAGATCTTCTTTTTTAGAGCGCCTTGTAGGCCTTTATCCATGCTGGTTCTAGAGAAGGTGTTGTGACAAATTGCCCTTTCAGTGTGACAAATCACCCTCAAATGACAGTCCTGTCTGTGACAAATTGCCCTTAACCCTGTGACAAATTGCCCTCAGAAGAAGCTGTTTTTTCACAAAGTTATCCCTGCTTATTGACTCTTTTTTATTTAGTGTGACAATCTAAAAACTTGTCACACTTCACATGGATCTGTCATGGCGGAAACAGCGGTTATCAATCACAAGAAACGTAAAAATAGCCCGCGAATCGTCCAGTCAAACGACCTCACTGAGGCGGCATATAGTCTCTCCCGGGATCAAAAACGTATGCTGTATCTGTTCGTTGACCAGATCAGAAAATCTGATGGCACCCTACAGGAACATGACGGTATCTGCGAGATCCATGTTGCTAAATATGCTGAAATATTCGGATTGACCTCTGCGGAAGCCAGTAAGGATATACGGCAGGCATTGAAGAGTTTCGCGGGGAAGGAAGTGGTTTTTTATCGCCCTGAAGAGGATGCCGGCGATGAAAAAGGCTATGAATCTTTTCCTTGGTTTATCAAACGTGCGCACAGTCCATCCAGAGGGCTTTACAGTGTACATATCAACCCATATCTCATTCCCTTCTTTATCGGGTTACAGAACCGGTTTACGCAGTTTCGGCTTAGTGAAACAAAAGAAATCACCAATCCGTATGCCATGCGTTTATACGAATCCCTGTGTCAGTATCGTAAGCCGGATGGCTCAGGCATCGTCTCTCTGAAAATCGACTGGATCATAGAGCGTTACCAGCTGCCTCAAAGTTACCAGCGTATGCCTGACTTCCGCCGCCGCTTCCTGCAGGTCTGTGTTAATGAGATCAACAGCAGAACTCCAATGCGCCTCTCATACATTGAGAAAAAGAAAGGCCGCCAGACGACTCATATCGTATTTTCCTTCCGCGATATCACTTCCATGACGACAGGATAGTCTGAGGGTTATCTGTCACAGATTTGAGGGTGGTTCGTCACATTTGTTCTGACCTACTGAGGGTAATTTGTCACAGTTTTGCTGTTTCCTTCAGCCTGCATGGATTTTCTCATACTTTTTGAACTGTAATTTTTAAGGAAGCCAAATTTGAGGGCAGTTTGTCACAGTTGATTTCCTTCTCTTTCCCTTCGTCATGTGACCTGATATCGGGGGTTAGTTCGTCATCATTGATGAGGGTTGATTATCACAGTTTATTACTCTGAATTGGCTATCCGCGTGTGTACCTCTACCTGGAGTTTTTCCCACGGTGGATATTTCTTCTTGCGCTGAGCGTAAGAGCTATCTGACAGAACAGTTCTTCTTTGCTTCCTCGCCAGTTCGCTCGCTATGCTCGGTTACACGGCTGCGGCGAGCGGTCCTTTTCATCACGTGCTATAAAAATAATTATAATTTAAATTTTTTAATATAAATATATAAATTAAAAATAGAAAGTAAAAAAAGAAATTAAAGAAAAAATAGTTTTTGTTTTCCGAAGATGTAAAAGACTCTAGGGGGATCGCCAACAAATACTACCTTTTATCTTGCTCTTCCTGCTCTCAGGTATTAATGCCGAATTGTTTCATCTTGTCTGTGTAGAAGACCACACACGAAAATCCTGTGATTTTACATTTTACTTATCGTTAATCGAATGTATATCTATTTAATCTGCTTTTCTTGTCTAATAAATATATATGTAAAGTACGCTTTTTGTTGAAATTTTTTAAACCTTTGTTTATTTTTTTTTCTTCATTCCGTAACTCTTCTACCTTCTTTATTTACTTTCTAAAATCCAAATACAAAACATAAAAATAAATAAACACAGAGTAAATTCCCAAATTATTCCATCATTAAAAGATACGAGGCGCGTGTAAGTTACAGGCAAGCGATCCGTCCGGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAAATTCCCGTTTTAAGAGCTTGGTGAGCGCTAGGAGTCACTGCCAGGTATCGTTTGAACACGGCATTAGTCAGGGAAGTCATAACACAGTCCTTTCCCGCAATTTTCTTTTTCTATTACTCTTGGCCTCCTCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTTTTTCCACCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAATGAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCCTAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAACAGGCCACACAATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACCATATGATACATGCTCTGGCCAAAGTATACCTATGAATGTCAGTAAGTATGTATACGAACAGTATGATACTGAAGATGACAAGGTAATGCATCATTCTATACGTGTCATTCTGAACGAGGCGCGCTTTCCTTTTTTCTTTTTGCTTTTTCTTTTTTTTTCTCTTGAACTCGACGGATCTATGCGGTGTGAAATACCGCACAGATGCGTCGCTAGTGATAATAAGTGACTGAGGTATGTGCTCTTCTTATCTCCTTTTGTAGTGTTGCTCTTATTTTAAACAACTTTGCGGTTTTTTGATGACTTTGCGATTTTGTTGTTGCTTTGCAGTAAATTGCAAGATTTAATAAAAAAACGCAAAGCAATGATTAAAGGATGTTCAGAATGAAACTCATGGAAACACTTAACCAGTGCATAAACGCTGGTCATGAAATGACGAAGgCTATCGCCATTGCACAGTTTAATGATGACAGCCCGGAAGCGAGGAAAATAaCCCGGCGCTGGAGAaTAGgTGaAGCAGCGGATTTAGTtGGGGTTTCTTCTCAGGCTATCAGAGATGCCGAGAAAGCAGGGCGACTACCGCACCCGGATATGGAAATTCGAGGACGGGTTGAGCAACGTGTTGGTTATACAATTGAACAAATTAATCATATGCGTGATGTGTTTGGTACGCGATTGCGACGTGCTGAAGACGTATTTCCACCGGTGATCGGGGTTGCTGCCCATAAAGGTGGCGTTTACAAAACCTCAGTTTCTGTTCATCTTGCTCAGGATCTGGCTCTGAAGGGGCTACGTGTTTTGCTCGTGGAAGGTAACGACCCCCAGGGAACAGCCTCAATGTATCACGGATGGGTACCAGATCTTCATATTCATGCAGAAGACACTCTCCTGCCTTTCTATCTTGGGGAAAAGGACGATGTCACTTATGCAATAAAGCCCACTTGCTGGCCGGGGCTTGACATTATTCCTTCCTGTCTGGCTCTGCACCGTATTGAAACTGAGTTAATGGGCAAATTTGATGAAGGTAAACTGCCCACCGATCCACACCTGATGCTCCGACTGGCCATTGAAACTGTTGCTCATGACTATGATGTCATAGTTATTGACAGCGCGCCTAACCTGGGTATCGGCACGATTAATGTCGTATGTGCTGCTGATGTGCTGATTGTTCCCACGCCTGCTGAGTTGTTTGACTACACCTCCGCACTGCAGTTTTTCGATATGCTTCGTGATCTGCTCAAGAACGTTGATCTTAAAGGGTTCGAGCCTGATGTACGTATTTTGCTTACCAAATACAGCAATAGCAATGGCTCTCAGTCCCCGTGGATGGAGGAGCAAATTCGGGATGCCTGGGGAAGCATGGTTCTAAAAAATGTTGTACGTGAAACGGATGAAGTTGGTAAAGGTCAGATCCGGATGAGAACTGTTTTTGAACAGGCCATTGATCAACGCTCTTCAACTGGTGCCTGGAGAAATGCTCTTTCTATTTGGGAACCTGTCTGCAATGAAATTTTCGATCGTCTGATTAAACCACGCTGGGAGATTAGATAATGAAGCGTGCGCCTGTTATTCCAAAACATACGCTCAATACTCAACCGGTTGAAGATACTTCGTTATCGACACCAGCTGCCCCGATGGTGGATTCGTTAATTGCGCGCGTAGGAGTAATGGCTCGCGGTAATGCCATTACTTTGCCTGTATGTGGTCGGGATGTGAAGTTTACTCTTGAAGTGCTCCGGGGTGATAGTGTTGAGAAGACCTCTCGGGTATGGTCAGGTAATGAACGTGACCAGGAGCTGCTTACTGAGGACGCACTGGATGATCTCATCCCTTCTTTTCTACTGACTGGTCAACAGACACCGGCGTTCGGTCGAAGAGTATCTGGTGTCATAGAAATTGCCGATGGGAGTCGCCGTCGTAAAGCTGCTGCACTTACCGAAAGTGATTATCGTGTTCTGGTTGGCGAGCTGGATGATGAGCAGATGGCTGCATTATCCAGATTGGGTAACGATTATCGCCCAACAAGTGCTTATGAACGTGGTCAGCGTTATGCAAGCCGATTGCAGAATGAATTTGCTGGAAATATTTCTGCGCTGGCTGATGCGGAAAATATTTCACGTAAGATTATTACCCGCTGTATCAACACCGCCAAATTGCCTAAATCAGTTGTTGCTCTTTTTTCTCACCCCGGTGAACTATCTGCCCGGTCAGGTGATGCACTTCAAAAAGCCTTTACAGATAAAGAGGAATTACTTAAGCAGCAGGCATCTAACCTTCATGAGCAGAAAAAAGCTGGGGTGATATTTGAAGCTGAAGAAGTTATCACTCTTTTAACTTCTGTGCTTAAAACGTCATCTGCATCAAGAACTAGTTTAAGCTCACGACATCAGTTTGCTCCTGGAGCGACAGTATTGTATAAGGGCGATAAAATGGTGCTTAACCTGGACAGGTCTCGTGTTCCAACTGAGTGTATAGAGAAAATTGAGGCCATTCTTAAGGAACTTGAAAAGCCAGCACCCTGATGCGACCTCGTTTTAGTCTACGTTTATCTGTCTTTACTTAATGTCCTTTGTTACAGGCCAGAAAGCATAACTGGCCTGAATATTCTCTCTGGGCCCACTGTTCCACTTGTATCGTCGGTCTGATAATCAGACTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATAATCAGACTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCATGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGAACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGATCCCACTCGTGTTGTCGGTCTGATTATCGGTCTGGGACCACGGTCCCACTTGTATTGTCGATCAGACTATCAGCGTGAGACTACGATTCCATCAATGCCTGTCAAGGGCAAGTATTGACATGTCGTCGTAACCTGTAGAACGGAGTAACCTCGGTGTGCGGTTGTATGCCTGCTGTGGATTGCTGCTGTGTCCTGCTTATCCACAACATTTTGCGCACGGTTATGTGGACAAAATACCTGGTTACCCAGGCCGTGCCGGCACGTTAACCGGGCTGCATCCGATGCAAGTGTGTCGCTGTCGACGAGCTCGCGAGCTCGGACATGAGGTTGCCCCGTATTCAGTGTCGCTGATTTGTATTGTCTGAAGTTGTTTTTACGTTAAGTTGATGCAGATCAATTAATACGATACCTGCGTCATAATTGATTATTTGACGTGGTTTGATGGCCTCCACGCACGTTGTGATATGTAGATGATAATCATTATCACTTTACGGGTCCTTTCCGGTGATCCGACAGGTTACGGGGCGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATTTTCCGGTTTAAGGCGTTTCCGTTCTTCTTCGTCATAACTTAATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGAAACGACAGGTGCTGAAAGCGAGCTTTTTGGCCTCTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCCGAGCTCATCGCTAATAACTTCGTATAGCATACATTATACGAAGTTATTTTCGATGCGGCCGCAAGGGGTTCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCACACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTA
挑取单菌落置于含15 μL Lysis Buffer的PCR管中,轻轻吹打三次裂解酵母菌,转移5 μL至一个新的PCR管中,4℃备用,其余10 μL进行菌落PCR检测,筛选出阳性克隆,然后电转化至EPI300大肠杆菌感受态细胞中,进行大量复制。通过chloramphenicol抗性筛选阳性重组子,通过大提质粒提取试剂盒(Omega Bio-tek公司)提取获得携带S蛋白氨基酸S173→P和Y179→C突变位点猪δ冠状病毒突变体病毒rPDCoV的cDNA感染性克隆重组质粒,命名为pGF-rPDCoV。
实施例2:通过PCR获得共转录本PDCoV N蛋白的cDNA
针对PDCoV中N基因的编码区,利用软件Oligo 7.0设计特异性引物PDCoV-T7-N-F和PDCoV-polyT-N-R(见表1),在N基因5’端插入T7启动子序列,在3’端插入polyA尾序列。以PDCoV HNZK-02 cDNA为模板,进行PCR扩增,得到N基因片段。反应体系为:PDCoV cDNA 2 μL,上游引物(10 μM)和下游引物(10μM)各1.5 μL,KOD OneTMPCR Master Mix-Blue- 25μL,ddH2O补齐至50 μL。扩增程序为:95℃ 3 min;98℃ 10 s,55℃ 5 s,68℃ 15 s,循环35次;68℃ 5 min。
实施例3:猪δ冠状病毒突变体病毒rPDCoV cDNA感染性克隆pGF-rPDCoV的线性化及体外转录
以猪δ冠状病毒感染性克隆pGF-rPDCoV为模板,利用ASCⅠ限制性内切酶(NewEngland Biolabs (Beijing) LTD)对载体进行单酶切,得到线性化的pGF-rPDCoV,反应体系为:pGF-rPDCoV 1 μg,10×rCutSmartTMBuffer 5 μL,ASCⅠ限制性内切酶1 μL,加ddH2O至50 μL,37℃过夜酶切。
酶切后的质粒以及通过PCR获得的PDCoV N基因片段均通过酚抽提-乙醇沉淀法进行纯化,分别以纯化后的线性化质粒和PDCoV N基因为模板,利用Thermo Fisher公司的mMESSAGE mMACHINE™ T7 转录试剂盒进行体外转录,得到猪δ冠状病毒突变体病毒rPDCoV的全长mRNA和猪δ冠状病毒N基因的mRNA,反应体系为:(1)rPDCoV mRNA获取,线性化的pGF-rPDCoV cDNA 1~2μg,2×NTP/CAP 25 μL,GTP 7.5 μL,10×Reaction Buffer 5 μL,Enzymemix 5 μL,ddH2O补足至50μL;(2)N基因mRNA获取,N基因cDNA 1 μg,2×NTP/CAP 10 μL,GTP0.75 μL,10×Reaction Buffer 2 μL,Enzymemix 2 μL,ddH2O补足至20 μL。反应程序为:(1)rPDCoV mRNA获取,32℃孵育8 h;(2)N基因mRNA获取,37℃孵育3 h。孵育后加入1 μLDnase 37℃ 15 min孵育降解DNA。随后采用有机溶剂抽提法对mRNA进行纯化,纯化后直接进行后续试验或-80℃长期保存。
实施例4:猪δ冠状病毒突变体病毒rPDCoV的拯救与生物学特性鉴定
1. 突变体病毒rPDCoV的拯救与扩增
将LLC-PK1细胞接种于T75细胞培养瓶中,待细胞密度达到90%左右,按照常规电转方法进行mRNA转染,参数为450 V 50 μF电转三次,间隔2 s。转染的rPDCoV mRNA与PDCoV N基因mRNA的质量均为20 μg,转染12 h后,将细胞培养液换成无血清含有trypsin(5 μg/mL,Gibco)的MEM培养基。
待出现明显细胞病变(细胞变大、变圆和脱落)时,收取细胞上清,获得拯救的突变体病毒rPDCoV。将拯救病毒在LLC-PK1细胞上连续传代,每一代培养物均置-80℃保存,取P5代的重组病毒进行后续试验。
2. 突变体病毒rPDCoV的鉴定
2.1间接免疫荧光检测病毒N蛋白表达
将弃掉上清的细胞用4%的多聚甲醛室温固定30 min,加入0.1%Triton-X100覆盖细胞,室温孵育10 min,弃去0.1% Triton-X 100后加入5% BSA覆盖细胞37℃封闭2 h,随后加入抗PDCoV N蛋白的单克隆抗体(实验室自制)4℃过夜孵育,1×PBST洗3次,加入GoatAnti-Mouse IgG (H&L) FITC二抗(Abmart)室温孵育1 h后,1×PBST洗3次,加入DAPI室温染色10 min,1×PBST洗3次,用荧光显微镜观察并记录实验结果(图4)。
2.2 RT-PCR鉴定
提取突变体病毒rPDCoV的RNA,用HiScript® II 1st Strand cDNA SynthesisKit (南京诺唯赞生物科技股份有限公司)反转录试剂盒获得重组病毒cDNA,分别使用引物PDCoV-A-F/PDCoV-A-R、PDCoV-B-F/PDCoV-B-R、PDCoV-C-F/PDCoV-C-R、PDCoV-D-F/PDCoV-D-R、PDCoV-E-F/PDCoV-E-R、PDCoV-F-F/PDCoV-F-R对六个片段进行RT-PCR扩增。
1%琼脂糖凝胶电泳后切胶回收,将胶回收后的核酸产物送至生工生物工程(上海)股份有限公司测序,利用软件Seqman将测序结果与原序列进行比对,结果显示:突变体病毒rPDCoV D片段上存在氨基酸S173→P和Y179→C突变,突变体病毒拯救成功。
3.生物学特性鉴定
3.1多步生长曲线
将LLC-PK1细胞接种至24孔板中,置于含5%CO2的37℃培养箱中培养,待细胞融合度达到100%进行感染。将rPDCoV和PDCoV HNZK-02以0.01 MOI分别感染LLC-PK1细胞。分别于感染后0 h、12 h、24 h、36 h、48 h和60 h,收集病毒上清,进行10倍梯度稀释,把待感染的LLC-PK1细胞用PBS洗2遍去除培养基中的血清,将稀释好的病毒液加入到96孔板中,每孔加入100 μL,各稀释度的病毒设8个重复,孵育2 h后弃去培养液,每孔加入100 μL含有trypsin(5 μg/mL)的MEM培养基继续在37℃培养。逐日观察并记录病变,于感染后4天观察细胞病变并按照Reed-Muench方法计算TCID50,绘制病毒的生长曲线。结果表明,突变体病毒rPDCoV在24 h时滴度达到最高,为1.01*109TCID50/mL,亲本病毒在36 h时达到最高,为1.75*106TCID50/mL,在感染细胞后的不同时间段内,突变体病毒rPDCoV的滴度均高于亲本病毒。
3.2突变体病毒感染仔猪空肠的IHC鉴定
取适量肠道组织,经脱水、置于10%福尔马林内固定24 h后取出,置于去离子水内漂洗两次后放入70%乙醇内,2h后取出并依次置于80%、90%及100%各浓度的乙醇中进行脱水;完成脱水过程后,将组织块放入无水乙醇和二甲苯按1:1比例混合的混合液中浸泡1.5h,而后转入二甲苯中泡至组织呈透明状态;取出组织呈透明状态的组织块,放入融化的石蜡内进行浸蜡,将浸蜡后的组织放入融化的固体石蜡中,带起完全凝固成含组织蜡块,即完成组织块的包埋过程;对包埋完毕的组织块进行修整后置于切片机上切成4~6μm的蜡带,将蜡片置于温水水面,蜡片展平后放于载玻片上在60 ℃温箱内烤片30 min;将干燥后的切片经二甲苯脱蜡后经不同浓度酒精逐渐脱苯,经蒸馏水复水后进行免疫组织化学(immunohistochemistry,IHC)染色,用PDCoV N蛋白单克隆抗体进行染色。采集的各组织经脱蜡、水化和流水冲洗后,用0.01 mol·L-1柠檬酸钠缓冲溶液(pH=6.0)对组织进行抗原修复,过氧化氢溶液室温孵育10 min阻断内源性过氧化物酶的干扰,3%BSA室温封闭30 min阻断抗体的非特异性结合。接着,将TGEVN蛋白单克隆抗体(1:500)在4℃条件下孵育过夜,生物素标记的羊抗小鼠IgG聚合物与样本室温孵育10 min,链霉素抗生素蛋白-过氧化物酶室温孵育10 min,最后用AEC显色试剂盒对样品进行显色。结果显示:突变体病毒rPDCoV感染组仔猪肠绒毛轻微萎缩,固有层与肠上皮间距无明显变化,黏膜上皮细胞中特异性红棕色着色点较少,与对照组对比不明显;PDCoV亲本病毒感染仔猪肠绒毛较突变体病毒感染仔猪萎缩严重,肠绒毛明显钝化、稀疏,并伴有较严重的肠壁变薄,同时,在黏膜上皮细胞中可见大量特异性红色着色点。表明突变体病毒rPDCoV在感染仔猪肠道中的致病力较亲本病毒减弱。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,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 (10)
1.一种猪δ冠状病毒突变体病毒rPDCoV,其特征在于:所述猪δ冠状病毒突变体病毒rPDCoV与PDCoV野毒S蛋白相比氨基酸突变位点为S173→P和Y179→C。
2.根据权利要求1所述的猪δ冠状病毒突变体病毒rPDCoV,其特征在于:所述猪δ冠状病毒突变体病毒rPDCoV的基因组序列如SEQ ID No.19所示。
3.一种猪δ冠状病毒突变体病毒rPDCoV感染性克隆的构建方法,其特征在于,步骤如下:
(1)以猪δ冠状病毒的反转录产物cDNA为模板,以序列如SEQ ID No.1- SEQ ID No.12所示的核苷酸片段为特异性引物,扩增猪δ冠状病毒的A-F片段并回收,获得A-F片段;
(2)以pGF载体为模板,以序列如SEQ ID No.13- SEQ ID No.16所示的核苷酸片段为引物,进行分段扩增并回收,获得pGF载体的G1片段和G2片段;
(3)向酿酒酵母VL6-48N菌液中加入藤黄节杆菌酶和β-巯基乙醇进行酶解反应,制备酵母感受态;
(4)将步骤(2)的G1片段和G2片段等比例混合,所得混合物与步骤(1)的A-F片段按1:1:1:1:1:1:1的质量比混合均匀,并转化至步骤(3)的酵母感受态中,转化后的酵母感受态经缺陷型固体培养基培养、筛选、扩培即得猪δ冠状病毒突变体病毒rPDCoV感染性克隆。
4.根据权利要求3所述的猪δ冠状病毒突变体病毒rPDCoV感染性克隆的构建方法,其特征在于:所述步骤(1)中猪δ冠状病毒为PDCoV HNZK-02毒株;获得的A-F片段中的A片段的5’端首位插入了T7启动子序列、F片段的3’端末端插入了ASCⅠ酶切位点序列。
5.根据权利要求3所述的猪δ冠状病毒突变体病毒rPDCoV感染性克隆的构建方法,其特征在于:所述步骤(2)中pGF载体的核苷酸序列如SEQ ID No.20所示。
6.根据权利要求5所述的猪δ冠状病毒突变体病毒rPDCoV感染性克隆的构建方法,其特征在于:所述步骤(3)中藤黄节杆菌酶的终浓度为0.15 U/μL 、β-巯基乙醇的终浓度为125μM/mL;所述步骤(4)中缺陷型固体培养基为组氨酸缺陷型固体培养基。
7.利用权利要求3-6任一项所述的方法构建的猪δ冠状病毒突变体病毒rPDCoVrPDCoV感染性克隆。
8.权利要求7所述的猪δ冠状病毒突变体病毒rPDCoVrPDCoV感染性克隆在病毒拯救中的应用。
9.根据权利要求8的应用,其特征在于,步骤为:
①猪δ冠状病毒突变体病毒rPDCoV cDNA感染性克隆经ASC Ⅰ内切酶酶切后,与猪δ冠状病毒N蛋白基因进行体外转录,得到猪δ冠状病毒突变体病毒rPDCoV的全长mRNA和猪δ冠状病毒N蛋白的全长mRNA;
②将猪δ冠状病毒突变体病毒rPDCoV的全长mRNA和猪δ冠状病毒N蛋白的mRNA等质量比混合后共转染至LLC-PK1细胞,当出现典型病变时收集病毒上清液获得拯救病毒,即猪δ冠状病毒突变体病毒rPDCoV。
10.权利要求1所述的猪δ冠状病毒突变体病毒rPDCoV在制备预防猪δ冠状病毒新型疫苗中的应用。
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