CN111434351B - TgCPC1在制备抗贫血药物中的应用 - Google Patents
TgCPC1在制备抗贫血药物中的应用 Download PDFInfo
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- CN111434351B CN111434351B CN201910026955.7A CN201910026955A CN111434351B CN 111434351 B CN111434351 B CN 111434351B CN 201910026955 A CN201910026955 A CN 201910026955A CN 111434351 B CN111434351 B CN 111434351B
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Abstract
本发明公开了TgCPC1在制备抗贫血药物中的应用。本发明是基于本发明发明人首次发现转染TgCPC1能促进人促红细胞生成素的表达得到的成果。本发明发明人发现转染TgCPC1质粒可以促进人促红细胞生成素的表达，这揭示了TgCPC1对促进人促红细胞生成素的表达的促进作用，为将TgCPC1用于制备抗贫血药物奠定了必要的基础。
Description
技术领域
本发明涉及生物医药领域，特别涉及TgCPC1在制备抗贫血药物中的应用。
背景技术
贫血为肾衰竭、癌症和免疫疾病等患者最常见的并发症。这类患者贫血症的治疗方法主要包括输血、补铁和红细胞生成刺激剂药物治疗。红细胞的生成过程主要发生于骨髓，受到内源性促红细胞生成素的调节。人促红细胞生成素(EPO)制剂主要用于治疗慢性肾功能衰竭引起的贫血、癌症及骨髓衰竭导致的贫血、失血后贫血，它减少了贫血患者的输血次数。重组人促红细胞生成素(recombinant human erythropoietin,r Hu EPO)已成功用于治疗肾性贫血20余年，但由于其半衰期相对较短，需要频繁注射给药，降低了病人的依从性。因此，寻找新的刺激EPO生成的药物很有必要。
发明内容
本发明的目的在于克服现有技术的缺点与不足，提供TgCPC1在制备抗贫血药物中的应用。
本发明的目的通过下述技术方案实现：TgCPC1在制备抗贫血的药物中的应用，是基于本发明发明人首次发现弓形虫TgCPC1能上调人的促红细胞生成素(EPO)所作出的发明创造。
所述的TgCPC1的氨基酸序列如下：
MEGMGSLRRRCAALPLVAGFLVFLGSIGVKADLPIHATVHDIKGDWTFYLSPAVPGDVSNCGSPSPNTNTSNLREELKDYKSFLSTQGGIKKELRVSLTDIPVSFATTRTAAALENPHRRTWKTLGVFDGQDKRKLVGSWTTVYDEGFEVDVGGRMRLMGFMKYNPQNNCSIVDGDLENSQGITDCYATDPTRTQIGWYIQQAEDGSRLSGCFYAEKPKAASSTPAAFVAIKETTRVEPASGALIGTDLKDSTSYISQSFVDTHNASPASSWRAGVNSVFANMNRRDLSRFVKDFGFKKMRPAGDSADGLSFMQSSTDISTADGDAASTQVYACPCKKGERSLDTRKLDESEQTLEVLSPVSALEVGHTNFLAAINPHVHESTATNGLDAQGTQAVAKAALSGAGATNTQEPLLLPKDFSWSDPFSNQAFDETVTNQGSCGSCYAVAATYALQKRFEIAASRMLGQEIRLFSAVQDEADGALSFLDLGSTSQLGELSAQSILSCSFYNQGCDGGFPYLVGKHARDIGVPQARCMEYQADHTQGCPFQKTASASESQSMLQADANAGACSEHARWYAKDYGYIGGCYECNHCSGEKQIMLEIYNNGPVPVAFDAPPSLFSYRSGVYDANSNHARVCDNDLPHHTGILTGWEYTNHAVTIVGWGETDGENGKPQKYWIVRNTWGPNWGVDGYVKIARGKNLGGIESQATFIDPDFSRGQGLKVAKAIEALKSKTM。
所述的TgCPC1的编码核苷酸序列如下所示：
ATGGAGGGTATGGGGTCGCTGCGCAGGCGGTGCGCTGCACTACCGCTCGTCGCGGGGTTCCTTGTTTTTCTGGGTTCGATTGGCGTAAAAGCGGATCTTCCCATTCACGCCACGGTACATGACATCAAGGGAGACTGGACGTTCTACTTGTCTCCAGCTGTACCCGGAGATGTTAGCAACTGCGGCTCCCCAAGCCCAAATACAAACACTTCGAATCTCCGCGAAGAACTCAAGGACTACAAGTCATTCTTGAGCACTCAAGGGGGAATCAAGAAGGAACTACGGGTGTCACTGACTGACATTCCAGTCTCGTTCGCAACTACTCGCACTGCCGCCGCACTCGAGAATCCCCACAGGCGCACCTGGAAAACGTTGGGTGTTTTCGATGGCCAGGATAAGCGGAAGCTTGTTGGCAGCTGGACAACGGTATACGACGAAGGTTTTGAGGTGGACGTTGGTGGCCGCATGAGGCTCATGGGGTTCATGAAATACAACCCGCAGAATAACTGCTCGATCGTCGACGGGGACTTGGAGAACTCTCAAGGAATCACAGACTGCTACGCAACCGACCCGACGCGAACGCAGATTGGTTGGTACATCCAGCAGGCAGAAGATGGCTCTCGTTTGTCAGGATGTTTCTATGCGGAGAAACCCAAGGCTGCAAGTTCAACTCCCGCCGCGTTCGTCGCGATCAAGGAAACCACGAGAGTTGAACCTGCTTCTGGTGCCTTGATTGGCACCGACCTGAAAGACTCAACTTCGTACATCTCTCAGTCGTTCGTGGATACACACAACGCGTCTCCTGCCTCAAGCTGGCGCGCGGGTGTCAACTCCGTTTTTGCGAACATGAACCGTCGAGATCTTTCTCGCTTTGTGAAGGATTTTGGCTTCAAAAAAATGCGACCTGCAGGTGACAGTGCCGATGGGCTTTCGTTCATGCAGTCTAGCACTGACATTTCAACTGCAGACGGAGACGCCGCCTCCACACAGGTGTATGCATGCCCGTGCAAGAAAGGCGAAAGGTCCCTAGACACGCGAAAGTTGGATGAGTCCGAACAGACTCTGGAGGTGCTGTCTCCTGTGAGCGCTCTGGAAGTTGGACACACAAACTTTTTGGCCGCAATCAATCCCCACGTTCACGAGAGCACAGCCACGAACGGACTCGACGCTCAAGGCACGCAAGCTGTGGCAAAAGCAGCACTGTCCGGCGCGGGTGCAACGAACACTCAGGAGCCCCTGCTACTCCCCAAAGACTTTTCGTGGTCGGATCCGTTCTCGAATCAGGCATTTGATGAGACTGTCACCAACCAAGGCAGCTGCGGGAGCTGCTATGCCGTGGCAGCAACCTACGCGCTGCAGAAGCGTTTCGAGATTGCAGCTTCTCGCATGCTTGGGCAGGAAATCCGACTTTTCAGCGCCGTCCAGGACGAGGCGGACGGAGCGTTGTCCTTTTTGGACCTTGGCAGTACGAGCCAACTCGGCGAGTTGTCTGCACAGTCAATCCTGTCTTGCTCCTTCTACAATCAAGGCTGCGACGGAGGCTTCCCGTACCTCGTGGGGAAACACGCGCGAGACATTGGTGTTCCGCAAGCCAGATGTATGGAGTACCAAGCTGACCACACACAAGGCTGCCCATTTCAAAAGACGGCTTCGGCCTCAGAGAGCCAATCGATGTTGCAGGCTGACGCGAATGCAGGAGCGTGTTCTGAGCACGCAAGATGGTATGCGAAGGACTACGGCTACATTGGCGGGTGCTACGAGTGCAATCACTGCTCTGGTGAAAAACAGATCATGTTGGAGATTTACAATAACGGTCCAGTGCCAGTTGCGTTCGATGCACCGCCTTCTCTCTTTTCCTACAGATCCGGGGTCTATGATGCGAACTCGAATCACGCGCGCGTGTGTGACAACGATTTGCCCCACCACACCGGAATTCTCACCGGGTGGGAATACACGAACCACGCAGTCACAATTGTGGGATGGGGAGAGACAGATGGTGAGAACGGAAAACCGCAGAAGTATTGGATTGTTCGGAACACGTGGGGTCCCAACTGGGGTGTCGACGGATACGTGAAGATTGCGCGCGGGAAGAACTTGGGGGGCATCGAAAGCCAAGCTACTTTCATCGACCCAGATTTCTCCAGAGGTCAAGGCTTGAAAGTGGCGAAAGCTATTGAAGCTCTGAAAAGTAAAACAATGTGA。
所述的抗贫血药物是含有TgCPC1蛋白和/或含有能表达TgCPC1蛋白的核酸的药物。
所述的核酸优选为DNA。
所述的抗贫血药物是能促进人促红细胞生成素生成的药物。
所述的贫血包括慢性肾功能衰竭引起的贫血、癌症及骨髓衰竭导致的贫血、失血后贫血。
本发明相对于现有技术具有如下的优点及效果：
本发明是基于本发明发明人首次发现转染TgCPC1能上调EPO得到的成果。本发明转染空质粒或TgCPC1至TLR4-HEK293T细胞中进行表达，转染24小时后，用LPS刺激24h后，提取两组细胞的RNA进行测序。RNAseq结果显示TgCPC1能上调EPO。后续用qPCR验证，发现TgCPC1确实能上调EPO的表达。这揭示了TgCPC1对EPO的上调作用，为将TgCPC1用于制备抗贫血药物奠定了必要的基础。
附图说明
图1是qPCR检测TgCPC1对EPO表达作用的结果图；其中，*P<0.05；**P<0.01；***P<0.001。
具体实施方式
下面结合实施例及附图对本发明作进一步的详细描述，但不用来限制本发明的范围。若未特别指出，实施例均参照常规实验条件，或者参照试剂盒制造厂商的说明书进行。实施例中所用到的工程菌、细胞株均为商业化的菌株或细胞株。
实施例1
RNA测序检测TgCPC1对TLR4-HEK293T细胞的mRNA表达水平的影响
(1)TgCPC1质粒的构建。
1)TgCPC1基因引物的设计。
用Omega软件设计扩增TgCPC1基因开放阅读框的引物，引物两端酶切位点分别为Not I和Kpn I，引物由广州艾基生物技术有限公司合成，引物的序列如下：
TgCPC1正向引物：5′-AAGGAAAAAAGCGGCCGCGATGGAGGGTATGGGGTCGCTGC-3′；
TgCPC1反向引物：5′-CGGGGTACCTCACATTGTTTTACTTTTCAGAGCTTCAATAGC-3′。
2)扩增TgCPC1基因。
用RNA抽提试剂盒(Omega)抽提弓形虫ME49株(弓形虫ME49株已在文献“Zhao YO,et al.Disruption of the Toxoplasma gondii parasitophorous vacuole byIFNgamma-inducible immunity-related GTPases(IRG proteins)triggers necroticcell death.PLoS pathogens.2009Feb；5(2):e1000288.PubMed PMID:19197351.PubmedCentral PMCID:2629126.”中公开)的总RNA，逆转录得到cDNA，以cDNA为模板扩增TgCPC1基因。
PCR扩增的反应体系如下：浓度为10μM的TgCPC1正向引物1μl、浓度为10μM的TgCPC1反向引物1μl、模板cDNA1μl(40ng)、5×Trans Start Fast Pfu Fly缓冲液1μl、Trans Start Fast Pfu Fly DNA聚合酶1μl(2.5U)、ddH2O补足至50μl。
PCR扩增的反应条件如下：95℃2min；95℃20s、65℃20s、72℃1min，30个循环；72℃5min。
3)TgCPC1基因的回收。
将上述PCR产物进行1％琼脂糖凝胶电泳，并用凝胶回收试剂盒(Omega)对PCR产物回收，实验步骤按试剂盒说明书，略有改动，步骤如下：
A、用手术刀片切下含有目的片段的琼脂糖胶，尽量切除DNA片段周围多余的琼脂糖胶以减少凝胶体积。
B、将凝胶块置于1.5ml微量离心管中。加入与凝胶等体积的结合缓冲液(Bindingbuffer(×p2))。将混合物置于50～55℃保温7min，直到凝胶完全溶解。
C、将HiBind DNA离心柱置于2ml收集管中，将700μl DNA/琼脂糖溶液加至HiBindDNA离心柱，室温下，10,000×g离心1min。
D、弃去液体，将HiBind DNA离心柱重新放回相同的收集管，向HiBind DNA离心柱中加入300μl结合缓冲液，室温下，10,000×g离心1min，洗HiBind DNA离心柱，弃去流出液并重新使用收集管。
E、加入700μl无水乙醇稀释的洗涤缓冲液洗涤HiBind DNA离心柱，室温下10,000×g离心1min。
F、弃去液体并最大转速空柱离心2min以干燥离心柱基膜。
G、将HiBind DNA离心柱置于干净的1.5ml离心管，向离心柱基膜中间滴加15～30μl洗脱缓冲液，室温放置1min后以13,000g离心1min洗脱DNA。
4)TgCPC1基因表达载体的构建。
将回收的TgCPC1片段和p3XFLAG-CMV-7.1表达载体(Sigma公司)经过Not I和KpnI双酶切处理，用T4DNA连接酶将双酶切后的TgCPC1DNA片段克隆至经双酶切的p3XFLAG-CMV-7.1表达载体中，然后转染大肠杆菌DH5α感受态细胞(全式金公司)，使用氨苄霉素对克隆进行筛选，将筛选得到的阳性克隆送去测序，DNA测序表明序列正确，命名为3×flagpCMV7.1-CPC1。
(2)转染
将人肾细胞系TLR4-HEK293T细胞(ATCC)接种到6孔板中，细胞量8×105个/孔，CO2恒温培养箱中培养24h后，待细胞密度在70％～80％时，用Lipo3000脂质体转染试剂盒转染。
设置如下试验组：
1)空白对照组：p3XFLAG-CMV-7.1空质粒(2μg/孔)；
2)实验组：p3XFLAG-CMV-7.1-CPC1质粒(2μg/孔)。
转染24小时后，加入脂多糖LPS(100ng/孔)刺激24小时。然后收集细胞，抽提RNA。
(3)RNA抽提
A、按照体积比50：1的比例往TRK裂解液(OMEGA)中加入β-巯基乙醇，在6孔板中，每孔加入700μl配好的TRK和β-巯基乙醇混合液。用移液枪反复抽吸，确保细胞裂解完全。
B、室温，10,000×g离心3分钟，将上清液转移到新的EP管中。
C、向裂解产物中加入等体积70％的乙醇，充分混匀。
D、将HiBind RNA离心柱置于2ml收集管中，将上述混合液加至HiBind RNA离心柱，室温下，10,000×g离心1min。
E、弃去液体，将HiBind RNA离心柱放入新的收集管中，向HiBind RNA离心柱中加入300μl RNA wash buffer I，室温下，10,000×g离心1min。
F、弃去液体，将HiBind RNA离心柱重新放回相同的收集管，向HiBind RNA离心柱中加入500μl RNA wash buffer I，室温下，10,000×g离心1min。
G、弃去液体，将HiBind RNA离心柱放入新的收集管中，向HiBind RNA离心柱中加入500μl RNA wash buffer II，室温下，10,000×g离心1min。弃去流出液并重新使用收集管。
H、加入500μl RNA wash buffer II，室温下，10,000×g离心1min。弃去流出液并重新使用收集管。
I、弃去液体并最大转速空柱离心2min以干燥离心柱基膜。
J、将HiBind RNA离心柱置于干净的1.5ml离心管，向离心柱基膜中间滴加30-50μlDEPC水，室温放置1min后以10,000g离心1min洗脱RNA。抽提好的RNA送华大基因进行RNA测序。表1展示的是RNA测序中的表达差异最大的前50个基因的情况。如表红色字体所示，与空白对照组相比，CPC1组EPO的表达量明显上升，且差异有统计学意义，说明CPC1可以促进EPO的表达。
表1RNA测序差异表达基因的表达情况表
实施例2
QPCR验证TgCPC1蛋白对EPO的上调作用。
(1)转染(同实施例1)
(2)RNA抽提(同实施例1)
(3)逆转录
逆转录的反应体系如下：5×primer scipt RT master mix 2μl、样品RNA 500ng、DEPC处理水补足至10μl。逆转录的反应条件如下：37℃15min，85℃5s。
(4)QPCR
所用QPCR引物如表2所示：
表2
| 基因 | 正向引物(5’-3’) | 反向引物(5’-3’) |
| β-actin | CACCATTGGCAATGAGCGGTTC | AGGTCTTTGCGGATGTCCACGT |
| EPO | GCATGTGGATAAAGCCGTCAGTG | GAGTTTGCGGAAAGTGTCAGCAG |
QPCR的反应体系：SYBR remix EX taq 12.5μl、浓度为10μM的PCR正向引物1μl、浓度为10μM的PCR反向引物1μl、DNA模板2μl(50ng)、蒸馏水补足至25μl。
QPCR的反应条件如下：95℃30s；95℃5s、60℃30s、65℃5s，40个循环。
实验结果如图1所示，与空质粒组相比，转染了TgCPC1实验组的EPO的表达明显升高。这验证了TgCPC1确实可以上调EPO的表达。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 暨南大学
<120> TgCPC1在制备抗贫血药物中的应用
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Val Ala Gly Phe Leu Val Phe Leu Gly Ser Ile Gly Val Lys Ala Asp
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Leu Pro Ile His Ala Thr Val His Asp Ile Lys Gly Asp Trp Thr Phe
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Tyr Leu Ser Pro Ala Val Pro Gly Asp Val Ser Asn Cys Gly Ser Pro
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Ser Pro Asn Thr Asn Thr Ser Asn Leu Arg Glu Glu Leu Lys Asp Tyr
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Lys Ser Phe Leu Ser Thr Gln Gly Gly Ile Lys Lys Glu Leu Arg Val
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Ser Leu Thr Asp Ile Pro Val Ser Phe Ala Thr Thr Arg Thr Ala Ala
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Ala Leu Glu Asn Pro His Arg Arg Thr Trp Lys Thr Leu Gly Val Phe
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Asp Gly Gln Asp Lys Arg Lys Leu Val Gly Ser Trp Thr Thr Val Tyr
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Asp Glu Gly Phe Glu Val Asp Val Gly Gly Arg Met Arg Leu Met Gly
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Phe Met Lys Tyr Asn Pro Gln Asn Asn Cys Ser Ile Val Asp Gly Asp
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Leu Glu Asn Ser Gln Gly Ile Thr Asp Cys Tyr Ala Thr Asp Pro Thr
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Arg Thr Gln Ile Gly Trp Tyr Ile Gln Gln Ala Glu Asp Gly Ser Arg
195 200 205
Leu Ser Gly Cys Phe Tyr Ala Glu Lys Pro Lys Ala Ala Ser Ser Thr
210 215 220
Pro Ala Ala Phe Val Ala Ile Lys Glu Thr Thr Arg Val Glu Pro Ala
225 230 235 240
Ser Gly Ala Leu Ile Gly Thr Asp Leu Lys Asp Ser Thr Ser Tyr Ile
245 250 255
Ser Gln Ser Phe Val Asp Thr His Asn Ala Ser Pro Ala Ser Ser Trp
260 265 270
Arg Ala Gly Val Asn Ser Val Phe Ala Asn Met Asn Arg Arg Asp Leu
275 280 285
Ser Arg Phe Val Lys Asp Phe Gly Phe Lys Lys Met Arg Pro Ala Gly
290 295 300
Asp Ser Ala Asp Gly Leu Ser Phe Met Gln Ser Ser Thr Asp Ile Ser
305 310 315 320
Thr Ala Asp Gly Asp Ala Ala Ser Thr Gln Val Tyr Ala Cys Pro Cys
325 330 335
Lys Lys Gly Glu Arg Ser Leu Asp Thr Arg Lys Leu Asp Glu Ser Glu
340 345 350
Gln Thr Leu Glu Val Leu Ser Pro Val Ser Ala Leu Glu Val Gly His
355 360 365
Thr Asn Phe Leu Ala Ala Ile Asn Pro His Val His Glu Ser Thr Ala
370 375 380
Thr Asn Gly Leu Asp Ala Gln Gly Thr Gln Ala Val Ala Lys Ala Ala
385 390 395 400
Leu Ser Gly Ala Gly Ala Thr Asn Thr Gln Glu Pro Leu Leu Leu Pro
405 410 415
Lys Asp Phe Ser Trp Ser Asp Pro Phe Ser Asn Gln Ala Phe Asp Glu
420 425 430
Thr Val Thr Asn Gln Gly Ser Cys Gly Ser Cys Tyr Ala Val Ala Ala
435 440 445
Thr Tyr Ala Leu Gln Lys Arg Phe Glu Ile Ala Ala Ser Arg Met Leu
450 455 460
Gly Gln Glu Ile Arg Leu Phe Ser Ala Val Gln Asp Glu Ala Asp Gly
465 470 475 480
Ala Leu Ser Phe Leu Asp Leu Gly Ser Thr Ser Gln Leu Gly Glu Leu
485 490 495
Ser Ala Gln Ser Ile Leu Ser Cys Ser Phe Tyr Asn Gln Gly Cys Asp
500 505 510
Gly Gly Phe Pro Tyr Leu Val Gly Lys His Ala Arg Asp Ile Gly Val
515 520 525
Pro Gln Ala Arg Cys Met Glu Tyr Gln Ala Asp His Thr Gln Gly Cys
530 535 540
Pro Phe Gln Lys Thr Ala Ser Ala Ser Glu Ser Gln Ser Met Leu Gln
545 550 555 560
Ala Asp Ala Asn Ala Gly Ala Cys Ser Glu His Ala Arg Trp Tyr Ala
565 570 575
Lys Asp Tyr Gly Tyr Ile Gly Gly Cys Tyr Glu Cys Asn His Cys Ser
580 585 590
Gly Glu Lys Gln Ile Met Leu Glu Ile Tyr Asn Asn Gly Pro Val Pro
595 600 605
Val Ala Phe Asp Ala Pro Pro Ser Leu Phe Ser Tyr Arg Ser Gly Val
610 615 620
Tyr Asp Ala Asn Ser Asn His Ala Arg Val Cys Asp Asn Asp Leu Pro
625 630 635 640
His His Thr Gly Ile Leu Thr Gly Trp Glu Tyr Thr Asn His Ala Val
645 650 655
Thr Ile Val Gly Trp Gly Glu Thr Asp Gly Glu Asn Gly Lys Pro Gln
660 665 670
Lys Tyr Trp Ile Val Arg Asn Thr Trp Gly Pro Asn Trp Gly Val Asp
675 680 685
Gly Tyr Val Lys Ile Ala Arg Gly Lys Asn Leu Gly Gly Ile Glu Ser
690 695 700
Gln Ala Thr Phe Ile Asp Pro Asp Phe Ser Arg Gly Gln Gly Leu Lys
705 710 715 720
Val Ala Lys Ala Ile Glu Ala Leu Lys Ser Lys Thr Met
725 730
<210> 2
<211> 2202
<212> DNA
<213> 弓形虫(Toxoplasma gondii)
<220>
<223> TgCPC1的编码核苷酸序列
<400> 2
atggagggta tggggtcgct gcgcaggcgg tgcgctgcac taccgctcgt cgcggggttc 60
cttgtttttc tgggttcgat tggcgtaaaa gcggatcttc ccattcacgc cacggtacat 120
gacatcaagg gagactggac gttctacttg tctccagctg tacccggaga tgttagcaac 180
tgcggctccc caagcccaaa tacaaacact tcgaatctcc gcgaagaact caaggactac 240
aagtcattct tgagcactca agggggaatc aagaaggaac tacgggtgtc actgactgac 300
attccagtct cgttcgcaac tactcgcact gccgccgcac tcgagaatcc ccacaggcgc 360
acctggaaaa cgttgggtgt tttcgatggc caggataagc ggaagcttgt tggcagctgg 420
acaacggtat acgacgaagg ttttgaggtg gacgttggtg gccgcatgag gctcatgggg 480
ttcatgaaat acaacccgca gaataactgc tcgatcgtcg acggggactt ggagaactct 540
caaggaatca cagactgcta cgcaaccgac ccgacgcgaa cgcagattgg ttggtacatc 600
cagcaggcag aagatggctc tcgtttgtca ggatgtttct atgcggagaa acccaaggct 660
gcaagttcaa ctcccgccgc gttcgtcgcg atcaaggaaa ccacgagagt tgaacctgct 720
tctggtgcct tgattggcac cgacctgaaa gactcaactt cgtacatctc tcagtcgttc 780
gtggatacac acaacgcgtc tcctgcctca agctggcgcg cgggtgtcaa ctccgttttt 840
gcgaacatga accgtcgaga tctttctcgc tttgtgaagg attttggctt caaaaaaatg 900
cgacctgcag gtgacagtgc cgatgggctt tcgttcatgc agtctagcac tgacatttca 960
actgcagacg gagacgccgc ctccacacag gtgtatgcat gcccgtgcaa gaaaggcgaa 1020
aggtccctag acacgcgaaa gttggatgag tccgaacaga ctctggaggt gctgtctcct 1080
gtgagcgctc tggaagttgg acacacaaac tttttggccg caatcaatcc ccacgttcac 1140
gagagcacag ccacgaacgg actcgacgct caaggcacgc aagctgtggc aaaagcagca 1200
ctgtccggcg cgggtgcaac gaacactcag gagcccctgc tactccccaa agacttttcg 1260
tggtcggatc cgttctcgaa tcaggcattt gatgagactg tcaccaacca aggcagctgc 1320
gggagctgct atgccgtggc agcaacctac gcgctgcaga agcgtttcga gattgcagct 1380
tctcgcatgc ttgggcagga aatccgactt ttcagcgccg tccaggacga ggcggacgga 1440
gcgttgtcct ttttggacct tggcagtacg agccaactcg gcgagttgtc tgcacagtca 1500
atcctgtctt gctccttcta caatcaaggc tgcgacggag gcttcccgta cctcgtgggg 1560
aaacacgcgc gagacattgg tgttccgcaa gccagatgta tggagtacca agctgaccac 1620
acacaaggct gcccatttca aaagacggct tcggcctcag agagccaatc gatgttgcag 1680
gctgacgcga atgcaggagc gtgttctgag cacgcaagat ggtatgcgaa ggactacggc 1740
tacattggcg ggtgctacga gtgcaatcac tgctctggtg aaaaacagat catgttggag 1800
atttacaata acggtccagt gccagttgcg ttcgatgcac cgccttctct cttttcctac 1860
agatccgggg tctatgatgc gaactcgaat cacgcgcgcg tgtgtgacaa cgatttgccc 1920
caccacaccg gaattctcac cgggtgggaa tacacgaacc acgcagtcac aattgtggga 1980
tggggagaga cagatggtga gaacggaaaa ccgcagaagt attggattgt tcggaacacg 2040
tggggtccca actggggtgt cgacggatac gtgaagattg cgcgcgggaa gaacttgggg 2100
ggcatcgaaa gccaagctac tttcatcgac ccagatttct ccagaggtca aggcttgaaa 2160
gtggcgaaag ctattgaagc tctgaaaagt aaaacaatgt ga 2202
<210> 3
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TgCPC1正向引物
<400> 3
aaggaaaaaa gcggccgcga tggagggtat ggggtcgctg c 41
<210> 4
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TgCPC1反向引物
<400> 4
cggggtacct cacattgttt tacttttcag agcttcaata gc 42
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> β-actin正向引物
<400> 5
caccattggc aatgagcggt tc 22
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> β-actin反向引物
<400> 6
aggtctttgc ggatgtccac gt 22
<210> 7
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> EPO正向引物
<400> 7
gcatgtggat aaagccgtca gtg 23
<210> 8
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> EPO反向引物
<400> 8
gagtttgcgg aaagtgtcag cag 23
Claims (4)
1.TgCPC1在制备通过促进人促红细胞生成素生成治疗贫血的药物中的应用，其特征在于：所述的TgCPC1的氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用，其特征在于：编码所述的TgCPC1的核苷酸序列如SEQ IDNO.2所示。
3.根据权利要求1所述的应用，其特征在于：所述的药物是含有TgCPC1蛋白和/或含有能表达TgCPC1蛋白的核酸的药物。
4.根据权利要求3所述的应用，其特征在于：所述的核酸为DNA。
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| Title |
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| DNA Vaccines Encoding Toxoplasma gondii Cathepsin C 1 Induce Protection against Toxoplasmosis in Mice;Yali Han等;《Korean J Parasitol》;20171031;第55卷(第5期);505-512 * |
| Toxoplasma gondii Cathepsin C1 inhibits NF-κB signalling through the positive regulation of the HIF-1α/EPO axis;Yumei Liu等;《Acta Tropica》;20190417;第195卷;35-43 * |
| 弓形虫CPC1蛋白对宿主NF-κB信号通路的抑制及其分子机制;刘宇美;《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》;20210215(第2期);E059-217 * |
| 弓形虫扁菱形蛋白酶4和组织蛋白酶C1的免疫学研究;韩亚莉;《中国优秀博硕士学位论文全文数据库(博士)医药卫生科技辑》;20181215(第12期);E059-73 * |
| 金东航主编.（五） 弓形虫病.《宠物防疫保健手册》.2015,141-144. * |
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