CN110951784A - 一种无标记猪β-防御素2基因定点敲入质粒载体及其应用 - Google Patents

一种无标记猪β-防御素2基因定点敲入质粒载体及其应用 Download PDF

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CN110951784A
CN110951784A CN201911385470.3A CN201911385470A CN110951784A CN 110951784 A CN110951784 A CN 110951784A CN 201911385470 A CN201911385470 A CN 201911385470A CN 110951784 A CN110951784 A CN 110951784A
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周锐
黎璐
黄靖
王安田
黄超
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Huazhong Agricultural University
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Abstract

本发明属于动物基因工程技术领域，具体涉及一种无标记猪β‑防御素2基因定点敲入质粒载体及其应用。本发明与转基因动物领域相关。构建了能表达双拷贝pbd‑2基因的供体质粒pcCAG‑R26‑PBD2‑T2A‑PBD2，该供体质粒还含有双侧带有loxP序列的新霉素抗性基因neoR表达盒。试验证明，将该质粒与能同时表达Cas9蛋白与靶向猪Rosa26位点的向导RNA的打靶质粒pX330‑pRosa26同时电转入受体细胞，实现了pbd‑2基因定点整合入猪的Rosa26位点，并通过Cre重组酶删除了抗性标记，经体细胞核移植及胚胎移植能够得到无抗性标记的pbd‑2基因定点敲入猪。

Description

一种无标记猪β-防御素2基因定点敲入质粒载体及其应用
技术领域
本发明属于转基因动物基因工程技术领域，具体涉及过表达猪β-防御素2(pbd-2)基因的定点敲入供体质粒pcCAG-R26-PBD2-T2A-PBD2及其构建方法。本发明的重组质粒可用于制备无标记pbd-2基因定点敲入细胞系与无标记pbd-2基因定点敲入转基因猪。
背景技术
防御素是一类阳离子抗菌肽，一般由18-45个氨基酸组成，含有6个半胱氨酸残基，形成3个分子内二硫键。在脊椎动物中，根据空间结构及二硫键结合能力，防御素被分为α、β和θ三大类。到目前为止，研究表明猪体内仅存在β防御素。通过与已知的猪β防御素-1进行序列比对，猪β-防御素2(PBD-2)于2006年首次被发现(Sang et al.2006)，被证实广泛分布于猪的心、肝、脾、肺、肾、小肠和大肠等各个组织中(Bao et al.2015)。
体外杀菌实验证明PBD-2对鼠伤寒沙门氏菌、李斯特菌、葡萄球菌、大肠杆菌、猪链球菌和猪红斑丹毒丝菌等数种猪病原菌均有显著抑制作用(Peng et al.2014；Veldhuizenet al.2008)。在抗病毒方面，PBD-2能显著抑制猪繁殖和呼吸综合征病毒在MA-104细胞中的增殖(Veldhuizen et al.2008)。分子对接结果显示PBD-2能与E型肝炎病毒的衣壳蛋白结合从而抑制该病毒(Quintero-Gil et al.2017)。利用显微注射法制备的PBD-2转基因小鼠对伪狂犬病毒表现出显著的抗病性(齐燕华，2016)。不仅如此，通过将PBD-2基因在猪体内过表达，猪对胸膜肺炎放线杆菌、猪流感病毒及副猪嗜血杆菌的抵抗力得到了显著的提高(Yang et al.2015；周锐et al.2019a；周锐et al.2019b)。在免疫调节方面，PBD-2能与TLR4结合从而抑制下游NF-κB的激活，减少炎性因子的释放从而抑制炎症(Huang etal.2019)。另外，将PBD-2作为饲料添加剂能促进猪的生长并缓解仔猪腹泻，同时还能改善猪的肠道微生物组成(Peng et al.2016)。因此，提高PBD-2在猪体内的表达量能提高猪对疾病的抵抗力。
目前，利用显微注射法、病毒载体法、转座子插入法等传统的转基因技术能实现目的基因在基因组中的随机整合，但这种随机整合也给转基因生物安全带来了不确定性，其中存在的问题包括转基因操作中基因插入的拷贝数与插入位点是否明确，外源基因表达是否稳定，是否包含标记基因等问题。而基因定点编辑技术则能有效解决上述问题，有利于转基因动物的遗传分析与监测。在猪的13号染色体上存在名为Rosa26的安全位点，该位点具有一个内源性启动子，驱动表达一种全身广泛存在的非编码RNA，当外源基因插入该位点时，如果外源基因的启动子与Rosa26位点的启动子方向相反，外源性的启动子能更稳定且高效地启动目的基因的表达，因此Rosa26位点被认为是理想的基因敲入位点之一。
CRISPR/Cas9系统是存在于细菌和古菌中的一种获得性免疫系统，被用于抵抗噬菌体侵染和质粒转移等。Cas9蛋白为一种核酸内切酶，向导RNA(简称gRNA)是一段与目的序列互补的RNA片段。Cas9蛋白在gRNA的引导下能靶向目的序列引起DNA的双链断裂，同时细胞开始自我修复，在含有同源臂的供体DNA的存在下，通过同源修复，能实现目的基因定点敲入的目的。
发明内容
本发明的目的在于提供一种构建无标记pbd-2基因定点敲入猪的供体质粒载体的方法，并利用所构建的重组质粒将pbd-2基因定点敲入大白猪的基因组中，应用于猪的抗病性状改良。
本发明的技术方案如下所述：
本发明构建了一个无标记pbd-2基因定点敲入大白猪的供体质粒，含有双拷贝的pbd-2基因及可删除的抗性标记neoR基因，被命名为pcCAG-R26-PBD2-T2A-PBD2质粒，该质粒的核苷酸序列如SEQ ID NO：4所示。该质粒与本实验室同时构建的能同时表达Cas9蛋白(GenBank：AST09977.1)与靶向猪Rosa26位点(SEQ ID NO：5第250-269位所述碱基序列)的向导RNA的打靶质粒pX330-pRosa26(核苷酸序列如SEQ ID NO：5所示)同时电转入受体细胞能实现pbd-2基因定点整合入猪的Rosa26位点，并通过Cre重组酶删除抗性标记neoR。
上述质粒pcCAG-R26-PBD2-T2A-PBD2能够在DNA双链断裂后提供同源定向修复的供体DNA，主要添加元件包括Kozak sequence，由T2A串联的双拷贝pbd-2基因(Genbank：NM_214442.2)，靶位点左侧同源臂(序列见后)，靶位点右侧同源臂(序列见后)，两个分别处于新霉素抗性基因表达盒5’及3’端的loxP序列(序列见后)。
上述供体质粒pcCAG-R26-PBD2-T2A-PBD2为序列表SEQ ID NO：4所示的DNA片段，其中：SEQ ID NO：4所述序列的第6-902位为靶位点左侧同源臂序列；第2624-2629位为Kozaksequence序列；第2630-2836位为第一个pbd-2基因的编码区序列；第2846-2899位为T2A序列；第2900-3109位为第二个pbd-2基因的编码区序列；第3666-3699位为新霉素抗性基因表达盒5’端的loxP序列；第5400-5433位为新霉素抗性基因表达盒3’端的loxP序列；第5434-6193位为靶位点右侧同源臂序列。
本发明还提供了利用pcCAG-R26-PBD2-T2A-PBD2质粒制备过表达PBD-2转基因猪的方法，所述的方法包括如下步骤：利用上述pbd-2基因定点敲入质粒载体pcCAG-R26-PBD2-T2A-PBD2和打靶质粒pX330-pRosa26，使pbd-2基因能定点敲入到猪胚胎成纤维细胞；利用Cre重组酶删除pbd-2基因的定点敲入的猪胚胎成纤维细胞基因组中新霉素抗性标记neoR；再利用无抗性标记的pbd-2基因定点敲入的猪胚胎成纤维细胞，制备得到无标记的定点敲入pbd-2基因的转基因猪(品种为大白猪)，从而实现了猪体内pbd-2基因的过表达。
生物学验证证明，利用本发明构建的猪体内过表达pbd-2基因定点敲入质粒载体，可以将双拷贝的pbd-2基因定点整合进猪13号染色体的Rosa26位点，并能通过Cre重组酶删除新霉素标记基因，最终成功得到无标记pbd-2基因定点敲入的猪。
本发明的实施对培育具有良好抗病性能的转基因猪新品种具有重要意义。
附图说明
图1：本发明的供体质粒pcCAG-R26-PBD2-T2A-PBD2的结构示意图。
图2：本发明使用的打靶质粒pX330-pRosa26的结构示意图。
图3：本发明构建供体质粒pcCAG-R26-PBD2-T2A-PBD2技术流程图。
图4：本发明定点敲入pbd-2基因技术流程图。
图5：利用SalI对pcCAG-R26-PBD2-T2A-PBD2供体质粒进行酶切鉴定。附图标记说明：图4中，M表示DNA分子量标准，P表示pcCAG-R26-PBD2-T2A-PBD2供体质粒。
图6：Rosa26基因位点转pbd-2基因的胚胎成纤维细胞培养上清液杀菌活性鉴定图。附图标记说明：TG表示转基因细胞，WT表示非转基因细胞，A.pleuropneumoniae表示胸膜肺炎放线杆菌，S.suis表示猪链球菌，CFU表示菌落形成单位，*表示p＜0.05。
图7：无标记Rosa26位点定点整合转pbd-2基因大白猪的鉴定图。附图标记说明：图7中的A图为PCR鉴定图，其中：M表示DNA分子量标准，BC表示空白对照，PC表示pcCAG-R26-PBD2-T2A-PBD2阳性对照，编号为仔猪耳缺编号，从上到下依次为PBD-2片段、neoR片段、On-target片段和β-actin片段；大小分别为547bp、266bp、1235bp、169bp；PBD-2片段表示pbd-2基因整合入基因组；无neoR扩增片段表示已删除标记基因；On-target片段表示pbd-2基因定点整合入Rosa26位点；β-actin片段为阳性对照，目的是为了确定PCR所用基因组模板没有问题。图7中的B图为免疫细胞化学鉴定图，其中：TG表示无标记Rosa26位点定点整合pbd-2基因的大白猪；WT表示非转基因大白猪；FITC绿色荧光信号表示PBD-2表达；DAPI蓝色荧光信号表示细胞核。
具体实施方式
对序列表的说明：
SEQ ID NO：1是pcCAG-PBD2载体的核苷酸序列；
SEQ ID NO：2是本发明制备的pcCAG-PBD2-T2A-PBD2载体的核苷酸序列；
SEQ ID NO：3是本发明制备的pcCAG-PBD2-T2A-PBD2-RA载体的核苷酸序列；
SEQ ID NO：4是本发明制备的pcCAG-R26-PBD2-T2A-PBD2载体的核苷酸序列；
SEQ ID NO：5是本发明制备的pX330-pRosa26载体的核苷酸序列；
SEQ ID NO：6是本发明制备的PBD2-T2A-PBD2的氨基酸序列；
本发明所述实施方式，如无特别说明，均为本领域的常规方案，所述试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：pbd-2基因的定点敲入质粒载体的构建
1.构建pcCAG-R26-PBD2-T2A-PBD2供体质粒
以pcCAG-PBD2载体(Yang et al.2015)为模板通过延伸PCR用KOD-Plus-Neo高保真酶(东洋纺，上海)扩增5’端添加EcoRI酶切位点与Kozak sequence且3’端添加了BamHI酶切位点的pbd-2基因，同时扩增了5’端添加了BamHI酶切位点与T2A序列且3’端添加了XhoI酶切位点的pbd-2基因，而后将两个pbd-2基因通过BamHI酶切并连接，将该片段命名为PBD2-T2A-PBD2。
将pcCAG-PBD2载体与PBD2-T2A-PBD2用EcoRI和XhoI进行双酶切后相连，将获得序列正确的重组载体命名为pcCAG-PBD2-T2A-PBD2。
以NCBI数据库中猪Rosa26基因的参考序列(Genbank:NC_010455.5)65757727-65758626区域为所述靶位点的右侧同源臂序列，用KOD-Plus-Neo高保真酶从猪的基因组中扩增靶位点右侧同源臂序列并在其5’端添加SacI酶切位点与loxP序列，在其3’端添加BstZ17I酶切位点，将所得片段命名为loxP-RA。
以pcCAG-PBD2载体为模板用KOD-Plus-Neo高保真酶扩增新霉素抗性基因表达盒片段并在其5’端添加DraIII酶切位点与loxP序列，在其3’端添加SacI酶切位点，将所得片段命名为loxP-neoR。
将上述所得的loxP-neoR片段与loxP-RA片段用SacI酶切后连接，将得到的片段命名为loxP-neoR-loxP-RA。
将上述pcCAG-PBD2-T2A-PBD2载体与loxP-neoR-loxP-RA片段用DraIII与BstZ17I进行双酶切并连接，将获得的序列正确的重组载体命名为pcCAG-PBD2-T2A-PBD2-RA载体。
以NCBI数据库中猪Rosa26基因的参考序列(Genbank:NC_010455.5)65758627-65759526区域为所述靶位点的左侧同源臂序列，用KOD-Plus-Neo高保真酶从猪的基因组中扩增靶位点左侧同源臂序列，在其3’端添加SpeI酶切位点及该酶切位点右侧同源序列，在其5’端添加该酶切位点左侧同源序列，将得到的片段命名为LA-SpeI。
将上述pcCAG-PBD2-T2A-PBD2-RA用SpeI酶切，通过
Figure BDA0002343484570000051
MultiS OneStep Cloning Kit(南京诺唯赞生物科技有限公司)将LA-SpeI连入该质粒骨架，获得的序列正确的重组载体即为pcCAG-R26-PBD2-T2A-PBD2供体质粒载体。
利用SalI对pcCAG-R26-PBD2-T2A-PBD2供体质粒载体进行酶切鉴定，酶切产生的小片段大小为结果如图4，结果显示得到的正确的重组载体pcCAG-R26-PBD2-T2A-PBD2。其中，SEQ ID NO：4所述序列的第1-6位与第6201-6206为SalI的识别序列；第6-902位为靶位点左侧同源臂序列；第917-922位为SpeI识别序列；第2624-2629位为Kozak sequence序列；第2630-2836位为第一个pbd-2基因的编码区序列；第2846-2899位为T2A序列；第2900-3109位为第二个pbd-2基因的编码区序列；第3657-3665位为DraIII识别序列；第3666-3699位为新霉素抗性基因表达盒5’端的loxP序列；第5394-5399位为SacI识别序列；第5400-5433位为新霉素抗性基因表达盒3’端的loxP序列；第5434-6193位为靶位点右侧同源臂序列；第6194-6199位为BstZ17I识别序列。
序列表SEQ ID NO：4所述序列的第2630-3109位是由T2A连接的两个pbd-2基因的序列编码的蛋白质序列(SEQ ID NO：6)。其中，SEQ ID NO：6所述的序列的第73-90位为T2A肽，在蛋白质翻译过程中会从SEQ ID NO：3的第89位和第90位氨基酸之间断开，使得SEQ IDNO：6所示的蛋白质最终形成一个具有脯氨酸N-末端的PBD-2前体肽(位置在SEQ ID NO：6的第90-159位)以及一个具有20个氨基酸残基的C-末端的PBD-2前体肽(位置在SEQ ID NO：6的第1-89位)。
2.pX330-pRosa26打靶质粒的构建
合成上下游寡核苷酸5’-CACCGCTCCTTCTCGATTATGGGC-3’和5’-AAACGCCCATAATCGAGAAGGAGC-3’，用ddH2O溶解至100μM，分别取上下游互补序列溶液1μL加至8μL ddH2O中混匀，置于95℃变性10min，再置于65℃孵育30min后室温自然冷却1h，得到sgRNA的靶DNA双链寡核苷酸。用BbsI(纽英伦生物技术有限公司，北京)酶切pX330(Addgene：42230)，将得到的载体骨架与sgRNA的靶DNA双链寡核苷酸相连，将得到序列正确的重组载体命名为pX330-pRosa26，该载体能表达靶向猪13号染色体Rosa26位点的sgRNA与Cas9蛋白质，该质粒核苷酸序列如SEQ ID NO：5所述。
实施例2：无标记Rosa26基因位点转pbd-2基因大白猪的构建与鉴定
1.无标记Rosa26基因位点转pbd-2基因大白猪的构建
首先构建无标记Rosa26基因位点转pbd-2基因胚胎成纤维细胞，其流程见图3。当猪胚胎成纤维细胞在直径100mm细胞培养皿中汇合度至80％左右时将旧培养基弃去，用杜氏磷酸缓冲液(DPBS)漂洗两遍后，加入1mL的胰酶消化至细胞脱壁，加入少许完全培养基(杜氏改良Eagle培养基+10％胎牛血清+1％青-链霉素)终止消化，离心后弃上清并加入DPBS重悬，再次离心弃上清。
加入800μL电转缓冲液Gene Pulser Electroporation Buffer(伯乐生命医学产品有限公司，上海)重悬，按8μg/孔的供体质粒pcCAG-R26-PBD2-T2A-PBD2和8μg/孔的打靶质粒pX330-pRosa26混匀后加入至细胞悬液，在4个0.2cm厚度的电转杯Gene PulserElectroporation Cuvettes(伯乐生命医学产品有限公司，上海)中分别加入200μL细胞悬液，加入过程中勿产生气泡，用Gene Pulser Xcell电穿孔系统按170V，15ms参数设置，电转后加入至6孔板中培养。
电转后24h弃去旧培养基，用磷酸盐缓冲液(PBS，赛默飞世尔科技，上海)漂洗一遍后加入新鲜完全培养基。电转48h后弃去旧培养基，6孔板中细胞用PBS漂洗后用胰酶消化至完全脱壁，加入完全培养基终止消化，离心后弃上清，每孔细胞均分为四份分别接种至直径100mm细胞培养皿中。当传代至直径100mm细胞培养皿后48h，弃去旧培养基，加入G418(赛默飞世尔科技，上海)浓度为400μg/mL的完全培养基，每2-3天换液一次。
当出现细胞克隆点时，用黑色马克笔标记后弃掉培养基，经DPBS缓冲液漂洗3遍后利用200μL的移液器枪头刮取细胞转入48孔细胞培养板进行培养，细胞长满后取一半用于鉴定，剩余细胞冻存。
提取细胞基因组DNA，进行PCR鉴定。利用如下引物对1(5’-GCTGGTTGTTGTGCTGTCTCATCA-3’和5’-CCCTCTAGACTCGAGTCAGGGTCAGC-3’)鉴定pbd-2基因是否整合进细胞基因组中，若正确整合，能扩增到大小为547bp的DNA片段；再利用如下引物对2(5’-CTGGTTGTTGTGCTGTCTCATCA-3’和5’-CCTTCTATCGCCTTCTTGACGAGTT-3’)鉴定pbd-2基因是否定点整合至猪13号染色体Rosa26位点，若定点整合，能扩增到大小为1216bp的DNA片段。
选取PCR鉴定正确的阳性克隆细胞，通过复苏、培养并传代至6孔板中，待细胞长至汇合率达60％时，用终浓度为3M的TAT-Cre重组酶(购自默克密理博公司，德国)处理细胞3h后，弃去培养基并用DPBS漂洗3遍，加入完全培养基继续培养。待细胞长满后，部分用于鉴定细胞培养上清对猪胸膜肺炎放线杆菌及猪链球菌的杀菌活性，结果证明转基因细胞培养上清杀菌效果显著高于非转基因细胞培养上清(见图6)。转基因细胞的另一部分送往武汉维尔鑫动物种质资源开发有限公司进行体细胞核移植及胚胎移植操作。每头受体母猪移植250枚左右的重构胚，最终共有3头受体母猪成功受孕。
2.无标记Rosa26基因位点转pbd-2基因大白猪的鉴定
受体母猪采用自然分娩的方式分娩转基因克隆猪。出生后一周之内采样出生小猪的耳组织进行转基因猪的PCR鉴定。具体步骤是：利用上述引物对2鉴定pbd-2基因是否成功整合至小猪基因组中；再利用引物对3(5’-GCCCCATGGCTGACTAATTTTTTTT-3’和5’-CGATTGTCTGTTGTGCCCAGTC-3’)鉴定标记基因是否成功删除，若成功删除则无法扩增出大小为266bp的条带；再利用引物对4(5’-CTTCCTTTCTCGCCACGTTC-3’和5’-TCGGTAAATAGCAATCAACTCAG-3’)鉴定pbd-2基因是否成功定点整合至猪13号染色体Rosa26位点，且能扩增出1235bp的DNA片段。最后利用引物对5(5’-GCCTCTCGTCTTGCTTGTTTTAAA-3’和5’-AGCAAGTGAGGGCGTATCCAG-3’)能扩增出长169bp的β-actin基因的部分片段，作为阳性对照以确保PCR所用基因组模板没有问题。
取小猪耳缘组织剪碎加入培养基分离猪耳成纤维细胞，使细胞在玻璃盖玻片上生长至一定汇合率，室温下在丙酮(-20℃预冷)中孵育细胞10min，用PBS洗涤细胞3次，每次5分钟。用含有1％牛血清白蛋白(BSA)与22.52mg/mL甘氨酸的PBST(PBS+0.1％Tween 20)孵育细胞30分钟，封闭抗体的非特异性结合。室温下，用PBD-2鼠单克隆抗体(谭雯雯，2010)(1％BSA，PBST)在湿盒中孵育细胞1小时，倒出溶液，用PBS洗涤细胞3次，每次5分钟。室温下，用二抗FITC Goat Anti-Mouse IgG(H+L)(Abclonal，武汉)(1％BSA，PBST)避光孵育细胞1小时，倒出二抗溶液，用PBS避光洗涤细胞3次，每次5分钟。用DAPI(上海碧云天生物技术有限公司)于37℃下避光孵育细胞10min，倒出溶液，用PBS洗涤细胞3次后于荧光显微镜下观察，绿色荧光信号表示PBD-2在细胞内的表达，蓝色荧光信号表示细胞核。结果显示相较于野生型猪耳成纤维细胞，转基因型猪耳成纤维细胞内可见大量表达的PBD-2蛋白。
阴性猪和阳性猪的鉴定结果如图7所示。结果表明，利用本发明的方法成功得到了无标记Rosa26位点定点整合pbd-2基因的大白猪，且PBD-2蛋白在胞内过表达。
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2.Huang C,Yang X,Huang J,Liu X,Yang X,Jin H,Huang Q,Li L,Zhou R(2019)Porcine Beta-Defensin 2 Provides Protection Against Bacterial Infection by aDirect Bactericidal Activity and Alleviates Inflammation via InterferenceWith the TLR4/NF-kappaB Pathway.Front Immunol 10:1673 doi:10.3389/fimmu.2019.01673.
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序列表
<110> 华中农业大学
<120> 一种无标记猪β-防御素2基因定点敲入质粒载体及其构建方法
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6379
<212> DNA
<213> 猪(Sus scrofa)
<220>
<221> CDS
<222> (1725)..(1934)
<400> 1
gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240
atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420
atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480
gcgatggggg cggggggggg ggggccgcgc gccagccggg gcggggcggg gcgaggggcg 540
gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600
tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660
gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720
ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780
gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840
ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900
tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960
cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020
ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080
tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140
cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc 1200
gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260
ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320
gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380
gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440
tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500
cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560
acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620
gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680
acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attc atg tgg gcc ctc 1736
Met Trp Ala Leu
1
tgc ttg ctg ctg ctg act gtc tgc ctc ctc tct tcc cag ctg gct gca 1784
Cys Leu Leu Leu Leu Thr Val Cys Leu Leu Ser Ser Gln Leu Ala Ala
5 10 15 20
ggt att aac ctg ctt acg ggt ctt ggc cag agg tcc gac cac tac ata 1832
Gly Ile Asn Leu Leu Thr Gly Leu Gly Gln Arg Ser Asp His Tyr Ile
25 30 35
tgt gcc aag aaa ggg ggg acc tgc aac ttc tcc ccc tgc ccg ctc ttc 1880
Cys Ala Lys Lys Gly Gly Thr Cys Asn Phe Ser Pro Cys Pro Leu Phe
40 45 50
aac agg att gaa ggg acc tgt tac agt ggc aag gcc aag tgc tgc atc 1928
Asn Arg Ile Glu Gly Thr Cys Tyr Ser Gly Lys Ala Lys Cys Cys Ile
55 60 65
cgc tga ccctgactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac 1984
Arg
70
tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 2044
ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 2104
gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 2164
ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 2224
aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc 2284
gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 2344
tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 2404
tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 2464
tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 2524
gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 2584
ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 2644
aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag 2704
ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 2764
aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 2824
agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 2884
ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 2944
gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt 3004
ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat 3064
caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct 3124
ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc 3184
tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc 3244
gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc 3304
acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg 3364
ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag 3424
aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc 3484
ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt 3544
cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc 3604
gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc 3664
tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg 3724
ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag 3784
cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg 3844
cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg 3904
aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct 3964
tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc 4024
gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg 4084
gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 4144
ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct 4204
ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 4264
tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 4324
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 4384
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 4444
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 4504
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 4564
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 4624
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 4684
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 4744
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 4804
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 4864
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 4924
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 4984
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 5044
ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 5104
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 5164
gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5224
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 5284
tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 5344
ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 5404
gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 5464
tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 5524
cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 5584
ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 5644
gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 5704
caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 5764
gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 5824
ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 5884
tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 5944
caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 6004
tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 6064
cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 6124
ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 6184
aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 6244
tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 6304
gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 6364
gaaaagtgcc acctg 6379
<210> 2
<211> 6655
<212> DNA
<213> 猪(Sus scrofa)
<220>
<221> CDS
<222> (1731)..(2210)
<400> 2
gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240
atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420
atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480
gcgatggggg cggggggggg ggggccgcgc gccagccggg gcggggcggg gcgaggggcg 540
gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600
tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660
gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720
ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780
gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840
ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900
tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960
cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020
ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080
tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140
cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc 1200
gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260
ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320
gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380
gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440
tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500
cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560
acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620
gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680
acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcgccacc atg agg gcc 1739
Met Arg Ala
1
ctc tgc ttg ctg ctg ctg act gtc tgc ctc ctc tct tcc cag ctg gct 1787
Leu Cys Leu Leu Leu Leu Thr Val Cys Leu Leu Ser Ser Gln Leu Ala
5 10 15
gca ggt att aac ctg ctt acg ggt ctt ggc cag agg tcc gac cac tac 1835
Ala Gly Ile Asn Leu Leu Thr Gly Leu Gly Gln Arg Ser Asp His Tyr
20 25 30 35
ata tgt gcc aag aaa ggg ggg acc tgc aac ttc tcc ccc tgc ccg ctc 1883
Ile Cys Ala Lys Lys Gly Gly Thr Cys Asn Phe Ser Pro Cys Pro Leu
40 45 50
ttc aac agg att gaa ggg acc tgt tac agt ggc aag gcc aag tgc tgc 1931
Phe Asn Arg Ile Glu Gly Thr Cys Tyr Ser Gly Lys Ala Lys Cys Cys
55 60 65
atc cgc gga tcc gga gag ggc aga gga agt ctg cta aca tgc ggt gac 1979
Ile Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp
70 75 80
gtc gag gag aat cct ggc cca atg agg gcc ctc tgc ttg ctg ctg ctg 2027
Val Glu Glu Asn Pro Gly Pro Met Arg Ala Leu Cys Leu Leu Leu Leu
85 90 95
act gtc tgc ctc ctc tct tcc cag ctg gct gca ggt att aac ctg ctt 2075
Thr Val Cys Leu Leu Ser Ser Gln Leu Ala Ala Gly Ile Asn Leu Leu
100 105 110 115
acg ggt ctt ggc cag agg tcc gac cac tac ata tgt gcc aag aaa ggg 2123
Thr Gly Leu Gly Gln Arg Ser Asp His Tyr Ile Cys Ala Lys Lys Gly
120 125 130
ggg acc tgc aac ttc tcc ccc tgc ccg ctc ttc aac agg att gaa ggg 2171
Gly Thr Cys Asn Phe Ser Pro Cys Pro Leu Phe Asn Arg Ile Glu Gly
135 140 145
acc tgt tac agt ggc aag gcc aag tgc tgc atc cgc tga ccctgactcg 2220
Thr Cys Tyr Ser Gly Lys Ala Lys Cys Cys Ile Arg
150 155 160
agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 2280
catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 2340
tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 2400
tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 2460
ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg 2520
ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2580
gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2640
ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2700
tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 2760
gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 2820
ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 2880
ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 2940
aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc 3000
aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 3060
tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 3120
agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 3180
ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 3240
tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 3300
aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc 3360
gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 3420
gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 3480
gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 3540
tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 3600
agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 3660
ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 3720
tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 3780
acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 3840
ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat 3900
gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 3960
ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 4020
tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 4080
ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 4140
ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 4200
cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 4260
ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 4320
ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 4380
atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 4440
ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 4500
tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 4560
cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 4620
attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 4680
tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 4740
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 4800
gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 4860
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 4920
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 4980
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 5040
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 5100
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 5160
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 5220
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 5280
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 5340
actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 5400
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtttttt tgtttgcaag 5460
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 5520
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 5580
aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 5640
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 5700
atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 5760
cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 5820
gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 5880
gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 5940
tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 6000
tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 6060
tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 6120
aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 6180
atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 6240
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 6300
catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 6360
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 6420
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 6480
gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 6540
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 6600
tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctg 6655
<210> 3
<211> 7484
<212> DNA
<213> 猪(Sus scrofa)
<220>
<221> CDS
<222> (1731)..(2210)
<400> 3
gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240
atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420
atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480
gcgatggggg cggggggggg ggggccgcgc gccagccggg gcggggcggg gcgaggggcg 540
gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600
tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660
gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720
ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780
gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840
ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900
tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960
cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020
ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080
tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140
cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc 1200
gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260
ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320
gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380
gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440
tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500
cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560
acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620
gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680
acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcgccacc atg agg gcc 1739
Met Arg Ala
1
ctc tgc ttg ctg ctg ctg act gtc tgc ctc ctc tct tcc cag ctg gct 1787
Leu Cys Leu Leu Leu Leu Thr Val Cys Leu Leu Ser Ser Gln Leu Ala
5 10 15
gca ggt att aac ctg ctt acg ggt ctt ggc cag agg tcc gac cac tac 1835
Ala Gly Ile Asn Leu Leu Thr Gly Leu Gly Gln Arg Ser Asp His Tyr
20 25 30 35
ata tgt gcc aag aaa ggg ggg acc tgc aac ttc tcc ccc tgc ccg ctc 1883
Ile Cys Ala Lys Lys Gly Gly Thr Cys Asn Phe Ser Pro Cys Pro Leu
40 45 50
ttc aac agg att gaa ggg acc tgt tac agt ggc aag gcc aag tgc tgc 1931
Phe Asn Arg Ile Glu Gly Thr Cys Tyr Ser Gly Lys Ala Lys Cys Cys
55 60 65
atc cgc gga tcc gga gag ggc aga gga agt ctg cta aca tgc ggt gac 1979
Ile Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp
70 75 80
gtc gag gag aat cct ggc cca atg agg gcc ctc tgc ttg ctg ctg ctg 2027
Val Glu Glu Asn Pro Gly Pro Met Arg Ala Leu Cys Leu Leu Leu Leu
85 90 95
act gtc tgc ctc ctc tct tcc cag ctg gct gca ggt att aac ctg ctt 2075
Thr Val Cys Leu Leu Ser Ser Gln Leu Ala Ala Gly Ile Asn Leu Leu
100 105 110 115
acg ggt ctt ggc cag agg tcc gac cac tac ata tgt gcc aag aaa ggg 2123
Thr Gly Leu Gly Gln Arg Ser Asp His Tyr Ile Cys Ala Lys Lys Gly
120 125 130
ggg acc tgc aac ttc tcc ccc tgc ccg ctc ttc aac agg att gaa ggg 2171
Gly Thr Cys Asn Phe Ser Pro Cys Pro Leu Phe Asn Arg Ile Glu Gly
135 140 145
acc tgt tac agt ggc aag gcc aag tgc tgc atc cgc tga ccctgactcg 2220
Thr Cys Tyr Ser Gly Lys Ala Lys Cys Cys Ile Arg
150 155 160
agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 2280
catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 2340
tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 2400
tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 2460
ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg 2520
ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2580
gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2640
ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2700
tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 2760
gtagtgataa cttcgtataa tgtatgctat acgaagttat ggccatcgcc ctgatagacg 2820
gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 2880
ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 2940
tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt 3000
ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 3060
aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 3120
gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 3180
cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 3240
ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt 3300
gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca 3360
ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat 3420
tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac 3480
agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc 3540
tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc 3600
tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag 3660
cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc 3720
ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg 3780
atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc 3840
ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc 3900
cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga 3960
cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca 4020
tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg 4080
atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg 4140
ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg 4200
gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga 4260
ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg 4320
gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat 4380
tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 4440
tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcagagctc 4500
ataacttcgt ataatgtatg ctatacgaag ttatggcggg attcttttgc cctggcttaa 4560
cctgattctt gggcgttgtc ctgcagggga ttgagcaggt gtacgaggac gagcccaatt 4620
tctctatatt cccacagtct tgagtttgtg tcacaaaata attatagtgg ggtggagatg 4680
ggaaatgagt ccaggcaaca cctaagcctg attttatgca ttgagactgc gtgttattac 4740
taaagatctt tgtgtcgcaa tttcctgatg aagggagata ggttaaaaag cacggatcta 4800
ctgagtttta cagtcatccc atttgtagac ttttgctaca ccaccaaagt atagcatctg 4860
agattaaata ttaatctcca aaccttaggc cccctcactt gcatccttac ggtcagataa 4920
ctctcactca tactttaagc ccattttgtt tgttgtactt gctcatccag tcccagtccc 4980
attggctttc tcctcacctg ttttaggtag ccagcaagtc atgaaatcag ataagttcca 5040
ccaccaatta acactaccca tcttgagcat aggcccaaca gtgcatttat tcctcattta 5100
ctgatgttcg tgaatattta ccttgatttt catttttttc tttttcttaa gctgggattt 5160
tactcctgac cctattcaca gtcagatgat cttgactacc actgcgattg gacctgaggt 5220
tcagcaatac tcccctttat gtcttttgaa tacttttcaa taaatctgtt tgtattttca 5280
ttagttagta actggtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata 5340
gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 5400
cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 5460
ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 5520
acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 5580
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 5640
gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 5700
ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 5760
cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 5820
ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 5880
taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 5940
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 6000
ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 6060
aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 6120
tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 6180
agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 6240
ttgatccggc aaacaaacca ccgctggtag cggttttttt gtttgcaagc agcagattac 6300
gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 6360
gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 6420
ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 6480
ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 6540
tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 6600
accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 6660
atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 6720
cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 6780
tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 6840
tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 6900
gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 6960
agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 7020
aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 7080
gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 7140
tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 7200
gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 7260
tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 7320
aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 7380
catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 7440
acaaataggg gttccgcgca catttccccg aaaagtgcca cctg 7484
<210> 4
<211> 8383
<212> DNA
<213> 猪(Sus scrofa)
<220>
<221> CDS
<222> (2630)..(3109)
<400> 4
gtcgacagag ggaggagcgc ttcctgctct tctcttgtca ctgattggcc gcttctcctc 60
ccgccgtgtg tgaaaacaca aatggcgtgt tttggttgga gtaaagctcc tgtcagttac 120
agcctcggga gtgcgcagcc tcccaggaac tctcgcattg ccccctgggt gggtaggtag 180
gtggggtgga gagagctgca caggcgggcg ctgtcggcct cctgcggggg gaggggaggg 240
tcagtgaaag tggctcccgc gcgggcgtcc tgccaccctc ccctccgggg gagtcggttt 300
acccgccgcc tgctcggctt tggtatctga ttggctgctg aagtcctggg aacggcccct 360
tgttattggc ttgggtccca aatgagcgaa accactacgc gagtcggcag ggaggcggtc 420
tttggtacgg ccctccccga ggccagcgcc gcagtgtctg gcccctcgcc cctgcgcaac 480
gtggcaggaa gcgcgcgcag gaggcggggg cgggctgccg ggccgaggct tctgggtggt 540
ggtgactgcg gctccgccct gggcgtccgc cgcctgaagg acgagactag ctctctacct 600
gctctcggac ccgtgggggt ggggggtgga ggaaggagtg gggggtcggt cctgctggct 660
tgtgggtggg aggcgcatgt tctccaaaaa cccgcgcgag ctgcaatcct gagggagctg 720
cagtggagga ggcggagaga aggccgcacc cttctccgca gggggagggg agtgccgcaa 780
tacctttatg ggagttctct gctgcctcct tttcctaagg accgccctgg gcctagaaaa 840
atccctccct cccccgcgat ctcgtcatcg cctccatgtc agtttgctcc ttctcgatta 900
tggacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 960
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 1020
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 1080
gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact tggcagtaca 1140
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 1200
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 1260
attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc actctcccca 1320
tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag 1380
cgatgggggc gggggggggg gggccgcgcg ccagccgggg cggggcgggg cgaggggcgg 1440
ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt 1500
ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg 1560
ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc 1620
cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg 1680
ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct gcgtgaaagc 1740
cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg gtgcgtgcgt 1800
gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg tgagcgctgc 1860
gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg gccgggggcg 1920
gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt 1980
gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc ctgcaccccc 2040
ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg 2100
cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc 2160
cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg ccccggagcg ccggcggctg 2220
tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg 2280
acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg caccccctct 2340
agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc 2400
gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc cgcaggggga 2460
cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg 2520
ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa 2580
cgtgctggtt gttgtgctgt ctcatcattt tggcaaagaa ttcgccacc atg agg gcc 2638
Met Arg Ala
1
ctc tgc ttg ctg ctg ctg act gtc tgc ctc ctc tct tcc cag ctg gct 2686
Leu Cys Leu Leu Leu Leu Thr Val Cys Leu Leu Ser Ser Gln Leu Ala
5 10 15
gca ggt att aac ctg ctt acg ggt ctt ggc cag agg tcc gac cac tac 2734
Ala Gly Ile Asn Leu Leu Thr Gly Leu Gly Gln Arg Ser Asp His Tyr
20 25 30 35
ata tgt gcc aag aaa ggg ggg acc tgc aac ttc tcc ccc tgc ccg ctc 2782
Ile Cys Ala Lys Lys Gly Gly Thr Cys Asn Phe Ser Pro Cys Pro Leu
40 45 50
ttc aac agg att gaa ggg acc tgt tac agt ggc aag gcc aag tgc tgc 2830
Phe Asn Arg Ile Glu Gly Thr Cys Tyr Ser Gly Lys Ala Lys Cys Cys
55 60 65
atc cgc gga tcc gga gag ggc aga gga agt ctg cta aca tgc ggt gac 2878
Ile Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp
70 75 80
gtc gag gag aat cct ggc cca atg agg gcc ctc tgc ttg ctg ctg ctg 2926
Val Glu Glu Asn Pro Gly Pro Met Arg Ala Leu Cys Leu Leu Leu Leu
85 90 95
act gtc tgc ctc ctc tct tcc cag ctg gct gca ggt att aac ctg ctt 2974
Thr Val Cys Leu Leu Ser Ser Gln Leu Ala Ala Gly Ile Asn Leu Leu
100 105 110 115
acg ggt ctt ggc cag agg tcc gac cac tac ata tgt gcc aag aaa ggg 3022
Thr Gly Leu Gly Gln Arg Ser Asp His Tyr Ile Cys Ala Lys Lys Gly
120 125 130
ggg acc tgc aac ttc tcc ccc tgc ccg ctc ttc aac agg att gaa ggg 3070
Gly Thr Cys Asn Phe Ser Pro Cys Pro Leu Phe Asn Arg Ile Glu Gly
135 140 145
acc tgt tac agt ggc aag gcc aag tgc tgc atc cgc tga ccctgactcg 3119
Thr Cys Tyr Ser Gly Lys Ala Lys Cys Cys Ile Arg
150 155 160
agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 3179
catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 3239
tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 3299
tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 3359
ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg 3419
ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 3479
gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 3539
ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 3599
tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 3659
gtagtgataa cttcgtataa tgtatgctat acgaagttat ggccatcgcc ctgatagacg 3719
gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 3779
ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 3839
tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt 3899
ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 3959
aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 4019
gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 4079
cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 4139
ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt 4199
gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca 4259
ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat 4319
tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac 4379
agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc 4439
tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc 4499
tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag 4559
cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc 4619
ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg 4679
atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc 4739
ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc 4799
cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga 4859
cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca 4919
tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg 4979
atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg 5039
ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg 5099
gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga 5159
ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg 5219
gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat 5279
tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 5339
tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcagagctc 5399
ataacttcgt ataatgtatg ctatacgaag ttatggcggg attcttttgc cctggcttaa 5459
cctgattctt gggcgttgtc ctgcagggga ttgagcaggt gtacgaggac gagcccaatt 5519
tctctatatt cccacagtct tgagtttgtg tcacaaaata attatagtgg ggtggagatg 5579
ggaaatgagt ccaggcaaca cctaagcctg attttatgca ttgagactgc gtgttattac 5639
taaagatctt tgtgtcgcaa tttcctgatg aagggagata ggttaaaaag cacggatcta 5699
ctgagtttta cagtcatccc atttgtagac ttttgctaca ccaccaaagt atagcatctg 5759
agattaaata ttaatctcca aaccttaggc cccctcactt gcatccttac ggtcagataa 5819
ctctcactca tactttaagc ccattttgtt tgttgtactt gctcatccag tcccagtccc 5879
attggctttc tcctcacctg ttttaggtag ccagcaagtc atgaaatcag ataagttcca 5939
ccaccaatta acactaccca tcttgagcat aggcccaaca gtgcatttat tcctcattta 5999
ctgatgttcg tgaatattta ccttgatttt catttttttc tttttcttaa gctgggattt 6059
tactcctgac cctattcaca gtcagatgat cttgactacc actgcgattg gacctgaggt 6119
tcagcaatac tcccctttat gtcttttgaa tacttttcaa taaatctgtt tgtattttca 6179
ttagttagta actggtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata 6239
gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 6299
cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 6359
ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 6419
acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 6479
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 6539
gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 6599
ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 6659
cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 6719
ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 6779
taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 6839
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 6899
ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 6959
aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 7019
tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 7079
agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 7139
ttgatccggc aaacaaacca ccgctggtag cggttttttt gtttgcaagc agcagattac 7199
gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 7259
gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 7319
ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 7379
ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 7439
tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 7499
accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 7559
atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 7619
cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 7679
tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 7739
tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 7799
gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 7859
agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 7919
aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 7979
gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 8039
tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 8099
gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 8159
tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 8219
aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 8279
catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 8339
acaaataggg gttccgcgca catttccccg aaaagtgcca cctg 8383
<210> 5
<211> 8508
<212> DNA
<213> 猪(Sus scrofa)
<220>
<221> CDS
<222> (1373)..(5473)
<400> 5
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ctccttctcg attatgggcg ttttagagct agaaatagca agttaaaata 300
aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360
ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420
acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 480
gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 540
tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 600
gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 660
gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 720
taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 780
acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 840
gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900
gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 960
gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac 1020
gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1080
tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1140
agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1200
acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1260
taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1320
gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag cc gac aag 1378
Asp Lys
1
aag tac agc atc ggc ctg gac atc ggc acc aac tct gtg ggc tgg gcc 1426
Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala
5 10 15
gtg atc acc gac gag tac aag gtg ccc agc aag aaa ttc aag gtg ctg 1474
Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu
20 25 30
ggc aac acc gac cgg cac agc atc aag aag aac ctg atc gga gcc ctg 1522
Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu
35 40 45 50
ctg ttc gac agc ggc gaa aca gcc gag gcc acc cgg ctg aag aga acc 1570
Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr
55 60 65
gcc aga aga aga tac acc aga cgg aag aac cgg atc tgc tat ctg caa 1618
Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln
70 75 80
gag atc ttc agc aac gag atg gcc aag gtg gac gac agc ttc ttc cac 1666
Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His
85 90 95
aga ctg gaa gag tcc ttc ctg gtg gaa gag gat aag aag cac gag cgg 1714
Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg
100 105 110
cac ccc atc ttc ggc aac atc gtg gac gag gtg gcc tac cac gag aag 1762
His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys
115 120 125 130
tac ccc acc atc tac cac ctg aga aag aaa ctg gtg gac agc acc gac 1810
Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp
135 140 145
aag gcc gac ctg cgg ctg atc tat ctg gcc ctg gcc cac atg atc aag 1858
Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys
150 155 160
ttc cgg ggc cac ttc ctg atc gag ggc gac ctg aac ccc gac aac agc 1906
Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser
165 170 175
gac gtg gac aag ctg ttc atc cag ctg gtg cag acc tac aac cag ctg 1954
Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu
180 185 190
ttc gag gaa aac ccc atc aac gcc agc ggc gtg gac gcc aag gcc atc 2002
Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile
195 200 205 210
ctg tct gcc aga ctg agc aag agc aga cgg ctg gaa aat ctg atc gcc 2050
Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala
215 220 225
cag ctg ccc ggc gag aag aag aat ggc ctg ttc gga aac ctg att gcc 2098
Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala
230 235 240
ctg agc ctg ggc ctg acc ccc aac ttc aag agc aac ttc gac ctg gcc 2146
Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala
245 250 255
gag gat gcc aaa ctg cag ctg agc aag gac acc tac gac gac gac ctg 2194
Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu
260 265 270
gac aac ctg ctg gcc cag atc ggc gac cag tac gcc gac ctg ttt ctg 2242
Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu
275 280 285 290
gcc gcc aag aac ctg tcc gac gcc atc ctg ctg agc gac atc ctg aga 2290
Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg
295 300 305
gtg aac acc gag atc acc aag gcc ccc ctg agc gcc tct atg atc aag 2338
Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys
310 315 320
aga tac gac gag cac cac cag gac ctg acc ctg ctg aaa gct ctc gtg 2386
Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val
325 330 335
cgg cag cag ctg cct gag aag tac aaa gag att ttc ttc gac cag agc 2434
Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser
340 345 350
aag aac ggc tac gcc ggc tac att gac ggc gga gcc agc cag gaa gag 2482
Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu
355 360 365 370
ttc tac aag ttc atc aag ccc atc ctg gaa aag atg gac ggc acc gag 2530
Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu
375 380 385
gaa ctg ctc gtg aag ctg aac aga gag gac ctg ctg cgg aag cag cgg 2578
Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg
390 395 400
acc ttc gac aac ggc agc atc ccc cac cag atc cac ctg gga gag ctg 2626
Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu
405 410 415
cac gcc att ctg cgg cgg cag gaa gat ttt tac cca ttc ctg aag gac 2674
His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp
420 425 430
aac cgg gaa aag atc gag aag atc ctg acc ttc cgc atc ccc tac tac 2722
Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr
435 440 445 450
gtg ggc cct ctg gcc agg gga aac agc aga ttc gcc tgg atg acc aga 2770
Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg
455 460 465
aag agc gag gaa acc atc acc ccc tgg aac ttc gag gaa gtg gtg gac 2818
Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp
470 475 480
aag ggc gct tcc gcc cag agc ttc atc gag cgg atg acc aac ttc gat 2866
Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp
485 490 495
aag aac ctg ccc aac gag aag gtg ctg ccc aag cac agc ctg ctg tac 2914
Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr
500 505 510
gag tac ttc acc gtg tat aac gag ctg acc aaa gtg aaa tac gtg acc 2962
Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr
515 520 525 530
gag gga atg aga aag ccc gcc ttc ctg agc ggc gag cag aaa aag gcc 3010
Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala
535 540 545
atc gtg gac ctg ctg ttc aag acc aac cgg aaa gtg acc gtg aag cag 3058
Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln
550 555 560
ctg aaa gag gac tac ttc aag aaa atc gag tgc ttc gac tcc gtg gaa 3106
Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu
565 570 575
atc tcc ggc gtg gaa gat cgg ttc aac gcc tcc ctg ggc aca tac cac 3154
Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His
580 585 590
gat ctg ctg aaa att atc aag gac aag gac ttc ctg gac aat gag gaa 3202
Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu
595 600 605 610
aac gag gac att ctg gaa gat atc gtg ctg acc ctg aca ctg ttt gag 3250
Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
615 620 625
gac aga gag atg atc gag gaa cgg ctg aaa acc tat gcc cac ctg ttc 3298
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe
630 635 640
gac gac aaa gtg atg aag cag ctg aag cgg cgg aga tac acc ggc tgg 3346
Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp
645 650 655
ggc agg ctg agc cgg aag ctg atc aac ggc atc cgg gac aag cag tcc 3394
Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser
660 665 670
ggc aag aca atc ctg gat ttc ctg aag tcc gac ggc ttc gcc aac aga 3442
Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg
675 680 685 690
aac ttc atg cag ctg atc cac gac gac agc ctg acc ttt aaa gag gac 3490
Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp
695 700 705
atc cag aaa gcc cag gtg tcc ggc cag ggc gat agc ctg cac gag cac 3538
Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His
710 715 720
att gcc aat ctg gcc ggc agc ccc gcc att aag aag ggc atc ctg cag 3586
Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln
725 730 735
aca gtg aag gtg gtg gac gag ctc gtg aaa gtg atg ggc cgg cac aag 3634
Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys
740 745 750
ccc gag aac atc gtg atc gaa atg gcc aga gag aac cag acc acc cag 3682
Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln
755 760 765 770
aag gga cag aag aac agc cgc gag aga atg aag cgg atc gaa gag ggc 3730
Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly
775 780 785
atc aaa gag ctg ggc agc cag atc ctg aaa gaa cac ccc gtg gaa aac 3778
Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn
790 795 800
acc cag ctg cag aac gag aag ctg tac ctg tac tac ctg cag aat ggg 3826
Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly
805 810 815
cgg gat atg tac gtg gac cag gaa ctg gac atc aac cgg ctg tcc gac 3874
Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp
820 825 830
tac gat gtg gac cat atc gtg cct cag agc ttt ctg aag gac gac tcc 3922
Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser
835 840 845 850
atc gac aac aag gtg ctg acc aga agc gac aag aac cgg ggc aag agc 3970
Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
855 860 865
gac aac gtg ccc tcc gaa gag gtc gtg aag aag atg aag aac tac tgg 4018
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp
870 875 880
cgg cag ctg ctg aac gcc aag ctg att acc cag aga aag ttc gac aat 4066
Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn
885 890 895
ctg acc aag gcc gag aga ggc ggc ctg agc gaa ctg gat aag gcc ggc 4114
Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly
900 905 910
ttc atc aag aga cag ctg gtg gaa acc cgg cag atc aca aag cac gtg 4162
Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
915 920 925 930
gca cag atc ctg gac tcc cgg atg aac act aag tac gac gag aat gac 4210
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp
935 940 945
aag ctg atc cgg gaa gtg aaa gtg atc acc ctg aag tcc aag ctg gtg 4258
Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val
950 955 960
tcc gat ttc cgg aag gat ttc cag ttt tac aaa gtg cgc gag atc aac 4306
Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn
965 970 975
aac tac cac cac gcc cac gac gcc tac ctg aac gcc gtc gtg gga acc 4354
Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr
980 985 990
gcc ctg atc aaa aag tac cct aag ctg gaa agc gag ttc gtg tac ggc 4402
Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly
995 1000 1005 1010
gac tac aag gtg tac gac gtg cgg aag atg atc gcc aag agc gag cag 4450
Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln
1015 1020 1025
gaa atc ggc aag gct acc gcc aag tac ttc ttc tac agc aac atc atg 4498
Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met
1030 1035 1040
aac ttt ttc aag acc gag att acc ctg gcc aac ggc gag atc cgg aag 4546
Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys
1045 1050 1055
cgg cct ctg atc gag aca aac ggc gaa acc ggg gag atc gtg tgg gat 4594
Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp
1060 1065 1070
aag ggc cgg gat ttt gcc acc gtg cgg aaa gtg ctg agc atg ccc caa 4642
Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln
1075 1080 1085 1090
gtg aat atc gtg aaa aag acc gag gtg cag aca ggc ggc ttc agc aaa 4690
Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1095 1100 1105
gag tct atc ctg ccc aag agg aac agc gat aag ctg atc gcc aga aag 4738
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys
1110 1115 1120
aag gac tgg gac cct aag aag tac ggc ggc ttc gac agc ccc acc gtg 4786
Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val
1125 1130 1135
gcc tat tct gtg ctg gtg gtg gcc aaa gtg gaa aag ggc aag tcc aag 4834
Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys
1140 1145 1150
aaa ctg aag agt gtg aaa gag ctg ctg ggg atc acc atc atg gaa aga 4882
Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1155 1160 1165 1170
agc agc ttc gag aag aat ccc atc gac ttt ctg gaa gcc aag ggc tac 4930
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr
1175 1180 1185
aaa gaa gtg aaa aag gac ctg atc atc aag ctg cct aag tac tcc ctg 4978
Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
ttc gag ctg gaa aac ggc cgg aag aga atg ctg gcc tct gcc ggc gaa 5026
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
ctg cag aag gga aac gaa ctg gcc ctg ccc tcc aaa tat gtg aac ttc 5074
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe
1220 1225 1230
ctg tac ctg gcc agc cac tat gag aag ctg aag ggc tcc ccc gag gat 5122
Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp
1235 1240 1245 1250
aat gag cag aaa cag ctg ttt gtg gaa cag cac aag cac tac ctg gac 5170
Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp
1255 1260 1265
gag atc atc gag cag atc agc gag ttc tcc aag aga gtg atc ctg gcc 5218
Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala
1270 1275 1280
gac gct aat ctg gac aaa gtg ctg tcc gcc tac aac aag cac cgg gat 5266
Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp
1285 1290 1295
aag ccc atc aga gag cag gcc gag aat atc atc cac ctg ttt acc ctg 5314
Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu
1300 1305 1310
acc aat ctg gga gcc cct gcc gcc ttc aag tac ttt gac acc acc atc 5362
Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile
1315 1320 1325 1330
gac cgg aag agg tac acc agc acc aaa gag gtg ctg gac gcc acc ctg 5410
Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1335 1340 1345
atc cac cag agc atc acc ggc ctg tac gag aca cgg atc gac ctg tct 5458
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser
1350 1355 1360
cag ctg gga ggc gac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa 5513
Gln Leu Gly Gly Asp
1365
agaaaaagta agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 5573
gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 5633
tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 5693
tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca 5753
tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc 5813
tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 5873
ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct 5933
ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc 5993
tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 6053
gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 6113
ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 6173
cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc 6233
tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 6293
ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt 6353
ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 6413
tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat 6473
gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 6533
tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 6593
cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 6653
tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 6713
ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 6773
ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 6833
attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 6893
gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 6953
ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 7013
cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 7073
gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 7133
tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 7193
gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 7253
ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 7313
tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 7373
gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 7433
caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 7493
cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt 7553
atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 7613
gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 7673
attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 7733
cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 7793
atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 7853
tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 7913
ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 7973
ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 8033
cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 8093
gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 8153
gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 8213
acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 8273
gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 8333
agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 8393
tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 8453
agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt 8508
<210> 6
<211> 159
<212> PRT
<213> 猪(Sus scrofa)
<400> 6
Met Arg Ala Leu Cys Leu Leu Leu Leu Thr Val Cys Leu Leu Ser Ser
1 5 10 15
Gln Leu Ala Ala Gly Ile Asn Leu Leu Thr Gly Leu Gly Gln Arg Ser
20 25 30
Asp His Tyr Ile Cys Ala Lys Lys Gly Gly Thr Cys Asn Phe Ser Pro
35 40 45
Cys Pro Leu Phe Asn Arg Ile Glu Gly Thr Cys Tyr Ser Gly Lys Ala
50 55 60
Lys Cys Cys Ile Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr
65 70 75 80
Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Arg Ala Leu Cys Leu
85 90 95
Leu Leu Leu Thr Val Cys Leu Leu Ser Ser Gln Leu Ala Ala Gly Ile
100 105 110
Asn Leu Leu Thr Gly Leu Gly Gln Arg Ser Asp His Tyr Ile Cys Ala
115 120 125
Lys Lys Gly Gly Thr Cys Asn Phe Ser Pro Cys Pro Leu Phe Asn Arg
130 135 140
Ile Glu Gly Thr Cys Tyr Ser Gly Lys Ala Lys Cys Cys Ile Arg
145 150 155

Claims (3)

1.一种适用于培育无标记pbd-2基因定点敲入猪的载体pcCAG-R26-PBD2-T2A-PBD2，其特征在于，该载体通过以下步骤制备获得：
(1)以SEQ ID NO：1所示序列的起始载体pcCAG-PBD2为材料，用EcoRI和XhoI双酶切，切除该载体中登录号为NW_018085039.1的pbd-2基因；
(2)以SEQ ID NO：1所示序列的起始载体pcCAG-PBD2为材料，通过延伸PCR扩增出5’端添加EcoRI酶切位点与Kozak sequence且3’端添加BamHI酶切位点的pbd-2基因，同时扩增5’端添加BamHI酶切位点与T2A序列且3’端添加XhoI酶切位点的pbd-2基因，而后将两个pbd-2基因通过BamHI酶切并经T4连接酶连接，将所得的连接产物命名为PBD2-T2A-PBD2，即SEQ ID NO：2第1731-2210位所示的碱基序列；
(3)在步骤(1)中EcoRI和XhoI切除载体pbd-2基因的位置，即SEQ ID NO：1第1720-1941碱基位置上定向插入步骤(2)中所述的PBD2-T2A-PBD2基因片段，得到如SEQ ID NO：2所示序列的中间载体pcCAG-PBD2-T2A-PBD2；
(4)以猪基因组为模板经延伸PCR扩增靶位点右侧同源臂序列，将该序列命名为loxP-RA，即SEQ ID NO：3第4495-5300位所示的碱基序列，在该片段同源臂5’端添加SacI酶切位点与loxP序列的3’端添加BstZ17I酶切位点；
(5)以SEQ ID NO：1所示的起始载体pcCAG-PBD2为模板经延伸PCR扩增抗性基因，将所得产物命名为loxP-neoR，即SEQ ID NO：3第2758-4494位所示的碱基序列，在该片段新霉素抗性基因5’端添加DraIII酶切位点与loxP序列，其3’端添加有SacI酶切位点；
(6)将步骤(4)与步骤(5)所述DNA片段loxP-RA和loxP-neoR用SacI酶切后经T4连接酶连接，将所得的连接产物命名为loxP-neoR-loxP-RA，即SEQ ID NO：3第2758-5300位所述的碱基序列；
(7)将步骤(3)所述中间载体pcCAG-PBD2-T2A-PBD2与步骤(6)所述loxP-neoR-loxP-RA用DraIII与BstZ17I进行双酶切后经T4连接酶连接，将所得的连接产物命名为pcCAG-PBD2-T2A-PBD2-RA载体，即SEQ ID NO：3所示的序列；
(8)以猪基因组为模板经延伸PCR扩增靶位点左侧同源臂序列，将该产物命名为LA-SpeI，即SEQ ID NO：4第8368-938位所示的碱基序列，该片段5’端添加有步骤(7)所述载体pcCAG-PBD2-T2A-PBD2-RA上SpeI酶切位点左侧同源序列，3’端添加有SpeI酶切位点以及步骤(7)所述载体上该酶切位点右侧同源序列；
(9)将步骤(7)所述载体pcCAG-PBD2-T2A-PBD2-RA用SpeI酶切后，将步骤(8)所述DNA片段LA-SpeI经同源重组酶连接至酶切后质粒骨架上，得到无标记pbd-2基因定点敲入供体质粒pcCAG-R26-PBD2-T2A-PBD2；
(10)将步骤(9)质粒pcCAG-R26-PBD2-T2A-PBD2与能同时表达Cas9蛋白与靶向猪Rosa26位点，其序列如SEQ ID NO：5第250-269位所述，碱基序列的向导RNA的打靶质粒pX330-pRosa26同时电转入猪的细胞实现pbd-2基因定点整合入猪基因组的Rosa26位点，并通过Cre重组酶删除抗性标记基因neoR。
2.权利要求1所述的无标记pbd-2基因定点敲入转基因猪的质粒载体pcCAG-R26-PBD2-T2A-PBD2在制备无标记pbd-2基因定点敲入转基因细胞或动物中的应用。
3.权利要求1所述的无标记pbd-2基因定点敲入转基因猪的质粒载体pcCAG-R26-PBD2-T2A-PBD2的制备方法在制备无标记pbd-2基因定点敲入转基因细胞或动物中的应用。
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CN114958759A (zh) * 2021-02-23 2022-08-30 南京启真基因工程有限公司 一种肌萎缩侧索硬化症模型猪的构建方法及应用
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