CN107033235A - 一种人tCD109及其检测试剂盒在胰腺癌诊断中的应用 - Google Patents

一种人tCD109及其检测试剂盒在胰腺癌诊断中的应用 Download PDF

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CN107033235A
CN107033235A CN201710448477.XA CN201710448477A CN107033235A CN 107033235 A CN107033235 A CN 107033235A CN 201710448477 A CN201710448477 A CN 201710448477A CN 107033235 A CN107033235 A CN 107033235A
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姜国忠
王伟伟
李文才
李盼
张建营
汲振余
陈奎生
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Zhengzhou Jinpujia Gene Technology Co.,Ltd.
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Abstract

本发明公开了一种人tCD109及其检测试剂盒在胰腺癌诊断中的应用，所述的tCD109序列为人CD109蛋白的Val22‑Arg663。本发明研究发现PRSS3能裂解CD109，使截断的CD109(truncated CD109，tCD109)释放到血液中，因此通过高效液相色谱法鉴定截断的CD109(tCD109)的N端氨基酸序列，明确裂解位点，确定tCD109的序列。本发明利用胰腺癌患者外周血中存在CD109的裂解片段，收集胰腺癌患者血清标本，用酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)方法/试剂盒检测tCD109含量，分析tCD109含量与胰腺癌患者生存之间的关系。结果表明胰腺癌患者血清中tCD109检测可用于早期诊断及判断患者预后。本发明的试剂盒可用于其他同时表达PRSS3和CD109的组织来源癌症的检测。

Description

一种人tCD109及其检测试剂盒在胰腺癌诊断中的应用
技术领域
本发明公开了一种人tCD109及其检测试剂盒在胰腺癌诊断中的应用，属于基因工程生物技术领域。
背景技术
CD109最早是从人CD34+急性髓样白血病细胞系KGla和内皮细胞中分离并鉴定的，编码CD109的基因定位于人类第6号染色体长臂上，包含33个外显子，其开放阅读框含有4335个碱基。CD109是一种细胞表面抗原，为糖基化磷脂酰肌醇偶联的糖蛋白，属于含硫酯的α2巨球蛋白/补体C3、C4、C5超家族，也是TGF-β/Smads信号通路中的辅助受体。CD109的N末端含有21个氨基酸组成的信号肽，C末端包含一个糖基化磷脂酰肌醇锚定联结基序，第22个外显子含有硫酯特征序列PYGCGEQ(918-924)以及其它保守序列，含有17个潜在的N-糖基化位点。
目前，已在多种正常细胞中检测到CD109的表达，包括内皮细胞、激活的血小板和T淋巴细胞、造血干细胞中的CD34+细胞和祖细胞。CD109在乳腺、唾液腺、泪腺的肌上皮细胞和前列腺基底细胞和睾丸组织中高表达，而在这些腺体的导管、腺泡和分泌细胞中不表达。
CD109在食道、肺、喉、口腔、子宫颈鳞状细胞癌中表达上调，在肺鳞状细胞癌组织中高度表达，在一半以上的基底样乳腺癌中表达。其中，CD109高表达的口腔癌前病变恶变为鳞状细胞癌的风险较大，可预测癌前病变的恶变倾向。尿路上皮癌中CD109阳性率为69.9％，而在正常膀胱上皮细胞中CD109不表达。有报道，人类恶性黑色素瘤细胞系中CD109基因转录水平显著上调，免疫组化检测4例恶性黑色素瘤中有2例呈CD109高表达。
目前临床上胰腺癌的诊断所用的肿瘤标志物有糖链蛋白CA19-9、CA242、CA50、CEA、CA125、CA724、MMP-7、TIMP-1、VEGF、受体结合型癌抗原(RCAS1)、肿瘤特异性生长因子(TSGF)等，无论是特异性和敏感性都不高。目前CD109在胰腺癌中的作用研究较少。
发明内容
为了解决上述问题，本发明的目的是提供一种tCD109及其检测试剂盒在胰腺癌诊断中的应用。本发明利用胰腺癌高表达的PRSS3裂解CD109，并通过高效液相色谱法鉴定截断的CD109(tCD109)的N端氨基酸序列，确定tCD109的序列，再建立ELISA方法，检测胰腺癌和胰腺炎患者血清中tCD109的表达，结合预后数据确定tCD109可作为胰腺癌的预后指标。
为了实现上述目的，本发明所采用的技术方案是：
一种人tCD109，所述的tCD109序列为人CD109蛋白的Val22-Arg663。
所述的tCD109的氨基酸序列如SEQ ID NO.1所示。
一种人tCD109作为胰腺癌临床诊断的靶标。
一种人tCD109在制备用于胰腺癌临床诊断的试剂盒中的应用。
一种癌症诊断试剂盒，包括测试板，测试板上包被有CD109抗体，CD109抗体的抗原为人CD109蛋白Val22-Arg663。
所述的试剂盒还包括系列浓度的tCD109标准品、偶联HRP的抗人源CD109、显色剂和终止液。
所述的癌症为胰腺癌。
所述的试剂盒用于检测外周血中游离的tCD109。
所述的tCD109序列为人CD109蛋白的Val22-Arg663。
所述的tCD109的氨基酸序列如SEQ ID NO.1所示。
本发明的有益效果：
1、PRSS3(胰蛋白酶原3)在胰腺癌中高表达，与肿瘤细胞增殖、浸润、进展和预后密切相关，是胰腺癌转移和较差预后的生物标志物之一。本发明研究发现PRSS3能裂解CD109，使截断的CD109(truncated CD109，tCD109)释放到血液中，并明确了裂解位点。
2、本发明发现胰腺癌患者外周血中存在CD109的裂解片段，并收集胰腺癌患者血清标本，用酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)方法/试剂盒检测tCD109含量，分析tCD109含量与胰腺癌患者生存之间的关系。结果表明胰腺癌患者血清中tCD109检测可用于早期诊断及判断患者预后。
3、本发明的试剂盒可用于其他同时表达PRSS3和CD109的组织来源癌症的检测。
附图说明
图1为考马斯亮蓝染色显示CD109经PRSS3裂解后的片段。
图2为高效液相色谱法(HPLC)鉴定tCD109的N端结果。其中，(1)-(6)分别代表Val、Ala、Pro、Gly、Pro、Arg。
图3为CD109裂解脱落片段的氨基酸序列(框中)。
图4为CD109裂解脱落片段的cDNA序列(框中)。
图5为血清中tCD109含量与胰腺癌患者的生存关系。
具体实施方式
以下通过实例对本发明进一步说明，并不是限定本发明的保护范围，本领域的技术人员根据说明书可以对这些实施方案进行修改，只要不脱离本发明的精神和范围都可以得到类似或相同的结果，均在本发明的保护范围之内。下述实施例中所使用的实验方法如无特殊说明，均为本领域的常规方法，或按照制造厂商所建议的条件与实施步骤。
实施例1、tCD109序列鉴定
tCD109序列鉴定方法，包括以下步骤：
(1)脱落CD109生物素化、裂解和捕获。24孔板中正常培养生长至90-95％的人胰腺癌Patu8988s细胞单层细胞用冰冷的PBS充分洗涤，并在4℃条件下加300μl含有生物素-XX，SSE(终浓度为0.5mg/ml，Invitrogen)的PBS/4％DMSO的溶液进行标记30min。随后，洗涤细胞，用1ml DMEM无血清培养基培养，并用终浓度为200nM的活性PRSS3 37℃条件下处理4h，同时加入等体积的未含活性PRSS3的溶剂作为对照。收集条件培养基并使用StreptavidinAgarose Resin分离生物素化的蛋白质。蛋白质通过SDS-PAGE胶分离染色。
(2)脱落CD109氨基酸序列鉴定。经PRSS3处理后，在Patu8988s细胞的条件培养基中分离生物素化的蛋白质中，捕获产生了几种染色的带，其中PRSS3处理后出现三条带，但在未处理的对照中不存在(图1)。条带回收后，将PRSS3处理得到的裂解片段送至上海中科新生命生物科技有限公司进行N端氨基酸序列分析。N端氨基酸序列为NH2-Val22-Ala-Pro-Gly-Pro-Arg(图2)。经分析后明确PRSS3裂解CD109的位点在R663-F664之间。由此确定tCD109氨基酸序列为Val22-Arg663(图3)(SEQ ID NO.1)，tCD109氨基酸对应的cDNA序列见图4(SEQID NO.2)。
实施例2、人胰腺癌患者血清中tCD109检测方法及预后分析
2.1、检测方法
采用本发明的试剂盒，对人胰腺癌患者血清中tCD109进行ELISA检测，试剂盒主要包括包被有CD109抗体(抗原为氨基酸Val22-Arg663)(本发明采用的是市售的CD109(H-7)抗体，购自Santa Cruz biotechnology公司，货号是sc-365780)的测试板、1％BSA、tCD109标准品(用大肠杆菌常规制作方法表达纯化的蛋白质)、偶联HRP的抗人源CD109(抗原为氨基酸Val22-Ser1268)(购自R&D Systems公司，货号是AF4385)、显色剂(四甲基联苯胺溶液)和终止液(H2SO4)，具体步骤为：
(1)收集73例胰腺疾病患者，其中病理明确确诊的胰腺癌患者58例，胰腺患者15例，血清标本用于tCD109ELISA检测。所有的血清样品用1％BSA进行1：50稀释，再以100μl/孔的量，将稀释的样品和系列浓度的tCD109标准品分别加入CD109(H-7)抗体包被的测试板的各孔中，4℃孵育过夜。孵育之后，弃掉孔中液体并且用PBS缓冲液洗涤4次。加入100μl偶联HRP的抗人源CD109(抗原为氨基酸Val22-Ser1268)，4℃孵育1h。用PBS缓冲液洗涤5次，每孔加入100μl四甲基联苯胺溶液室温孵育30min。最后，每孔加入100μl 2M H2SO4终止液。反应终止后，使用酶标仪测量各个样品在450nm/570nm的吸光度，并根据标准曲线计算各个样品的浓度。
统计结果表明，在胰腺炎患者血清中，没有检测到tCD109(0/15)。而在58例经病理确诊的胰腺癌患者中，有38例患者检测血清中检测到tCD109，阳性率65.5％(38/58)，20例(34.5％，20/58)胰腺癌患者血清中未检测到tCD109。说明本发明检测游离的tCD109的方法用于胰腺癌诊断的特异性达到100％，而敏感性为65.5％。
2.2、预后分析
用Kaplan-Meier方法比较了58例血清中tCD109含量与胰腺癌患者的生存关系(图5)，血清中tCD109含量升高(tCD109+)的胰腺癌患者的平均生存时间(14个月)明显低于血清tCD109阴性(tCD109-)的患者的平均生存时间(18个月)(p<0.05)。为了明确其他因素是否影响血清中tCD109含量与生存的关系，进行了多因素分析。Cox回归分析(表1)显示，血清tCD109含量和远端转移是两个与胰腺癌患者生存相关的重要因素，结果说明，胰腺癌患者血清tCD109检测除了用于诊断，也是重要的预后指标。
表1、Cox回归分析结果
以上所述仅为本发明最佳的实施例，对于本领域的技术人员来说，本发明可以有各种更改和变化。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。
SEQUENCE LISTING
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gtgactattg gggtggagct tctggaacac tgcccttcac aggtgactgt gaaggcggag 120
ctgctcaaga cagcatcaaa cctcactgtc tctgtcctgg aagcagaagg agtctttgaa 180
aaaggctctt ttaagacact tactcttcca tcactacctc tgaacagtgc agatgagatt 240
tatgagctac gtgtaaccgg acgtacccag gatgagattt tattctctaa tagtacccgc 300
ttatcatttg agaccaagag aatatctgtc ttcattcaaa cagacaaggc cttatacaag 360
ccaaagcaag aagtgaagtt tcgcattgtt acactcttct cagattttaa gccttacaaa 420
acctctttaa acattctcat taaggacccc aaatcaaatt tgatccaaca gtggttgtca 480
caacaaagtg atcttggagt catttccaaa acttttcagc tatcttccca tccaatactt 540
ggtgactggt ctattcaagt tcaagtgaat gaccagacat actatcaatc atttcaggtt 600
tcagaatatg tattaccaaa atttgaagtg actttgcaga caccattata ttgttctatg 660
aattctaagc atttaaatgg taccatcacg gcaaagtata catatgggaa gccagtgaaa 720
ggagacgtaa cgcttacatt tttaccttta tccttttggg gaaagaagaa aaatattaca 780
aaaacattta agataaatgg atctgcaaac ttctctttta atgatgaaga gatgaaaaat 840
gtaatggatt cttcaaatgg actttctgaa tacctggatc tatcttcccc tggaccagta 900
gaaattttaa ccacagtgac agaatcagtt acaggtattt caagaaatgt aagcactaat 960
gtgttcttca agcaacatga ttacatcatt gagttttttg attatactac tgtcttgaag 1020
ccatctctca acttcacagc cactgtgaag gtaactcgtg ctgatggcaa ccaactgact 1080
cttgaagaaa gaagaaataa tgtagtcata acagtgacac agagaaacta tactgagtac 1140
tggagcggat ctaacagtgg aaatcagaaa atggaagctg ttcagaaaat aaattatact 1200
gtcccccaaa gtggaacttt taagattgaa ttcccaatcc tggaggattc cagtgagcta 1260
cagttgaagg cctatttcct tggtagtaaa agtagcatgg cagttcatag tctgtttaag 1320
tctcctagta agacatacat ccaactaaaa acaagagatg aaaatataaa ggtgggatcg 1380
ccttttgagt tggtggttag tggcaacaaa cgattgaagg agttaagcta tatggtagta 1440
tccaggggac agttggtggc tgtaggaaaa caaaattcaa caatgttctc tttaacacca 1500
gaaaattctt ggactccaaa agcctgtgta attgtgtatt atattgaaga tgatggggaa 1560
attataagtg atgttctaaa aattcctgtt cagcttgttt ttaaaaataa gataaagcta 1620
tattggagta aagtgaaagc tgaaccatct gagaaagtct ctcttaggat ctctgtgaca 1680
cagcctgact ccatagttgg gattgtagct gttgacaaaa gtgtgaatct gatgaatgcc 1740
tctaatgata ttacaatgga aaatgtggtc catgagttgg aactttataa cacaggatat 1800
tatttaggca tgttcatgaa ttcttttgca gtctttcagg aatgtggact ctgggtattg 1860
acagatgcaa acctcacgaa ggattatatt gatggtgttt atgacaatgc agaatatgct 1920
gagagg 1926

Claims (10)

1.一种人tCD109，其特征在于，所述的tCD109序列为人CD109蛋白的Val22-Arg663。
2.根据权利要求1所述的人tCD109，其特征在于，所述的tCD109的氨基酸序列如SEQ IDNO.1所示。
3.一种权利要求1或2所述的人tCD109作为胰腺癌临床诊断的靶标。
4.一种权利要求1或2所述的人tCD109在制备用于胰腺癌临床诊断的试剂盒中的应用。
5.一种癌症诊断试剂盒，其特征在于，所述的试剂盒包括测试板，测试板上包被有CD109抗体，CD109抗体的抗原为CD109蛋白的Val22-Arg663。
6.根据权利要求5所述的试剂盒，其特征在于，所述的试剂盒还包括系列浓度的tCD109标准品、偶联HRP的抗人源CD109、显色剂和终止液。
7.根据权利要求5或6所述的试剂盒，其特征在于，所述的癌症为胰腺癌。
8.根据权利要求7所述的试剂盒，其特征在于，所述的试剂盒用于检测外周血中游离的tCD109。
9.根据权利要求8所述的试剂盒，其特征在于，所述的tCD109序列为人CD109蛋白的Val22-Arg663。
10.根据权利要求9所述的试剂盒，其特征在于，所述的tCD109的氨基酸序列如SEQ IDNO.1所示。
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