CN103333861A - 特异性表达hla-g1抗原的细胞株k562 - Google Patents
特异性表达hla-g1抗原的细胞株k562 Download PDFInfo
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- CN103333861A CN103333861A CN2013102181312A CN201310218131A CN103333861A CN 103333861 A CN103333861 A CN 103333861A CN 2013102181312 A CN2013102181312 A CN 2013102181312A CN 201310218131 A CN201310218131 A CN 201310218131A CN 103333861 A CN103333861 A CN 103333861A
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Abstract
本发明公开了特异性表达HLA-G1抗原的细胞株K562，其保藏编号为CCTCC：C201351，保藏日期为2013年5月8日，保藏单位是中国典型培养物保藏中心。由该细胞株生产的HLA-G1分子量为39kDa，包括重链、轻链及抗原肽；其中重链包括α1、α2、α3结构域和跨膜区、胞内肽段，轻链即β2微球蛋白（β2m），分子量12kDa，由人类第15号染色体编码。该蛋白特异性强，稳定性好，具有很好的商业化应用价值。
Description
技术领域
本发明属于生物工程领域，特别涉及一种稳定表达膜结合型HLA-G1的肿瘤细胞。
背景技术
1987 年，Geraghty 等首次成功克隆人白细胞抗原-G（human leukocyte antigen-G, HLA-G）基因，该基因全长6.0kb ，位于人类第6 号染色体短臂远侧6p21.3。HLA-G初始转录产物经选择性剪接后产生7种成熟mRNA，分别编码7种蛋白质异构体分子，包含四种膜结合型HLA-G分子（HLA-G1、-G2、-G3、-G4）和三种可溶性HLA-G分子（HLA-G5、-G6、-G7）（见图1）。HLA-G1～-G7 异构体分子的分子量分别为39kD, 31kD, 23kD, 30kD, 37kD, 27kD及17kD。HLA-G抗原在生理情况下仅限制性表达在少数免疫豁免组织，如胸腺、角膜、胰岛及间充质干细胞等，但在病理情况下，HLA-G分子能在多种恶性肿瘤微环境及器官移植术后、病毒感染等病理组织中诱导性表达，与疾病的发生及进展密切相关。
HLA-G与其受体结合后，能够诱导免疫耐受，具有免疫抑制功能。目前已发现能够识别结合HLA-G分子的免疫球蛋白样受体共有三种：免疫球蛋白样转录物-2（immunoglobulin-like transcript-2, ILT2/CD85j）、免疫球蛋白样转录物-4（immunoglobulin-like transcript-4, ILT4/CD85d）及杀伤细胞免疫球蛋白样受体（killer cell immunoglobulin-like receptor, KIR2DL4/CD158d）。ILT2表达在所有的单核、树突状细胞和B细胞，以及部分T细胞和NK细胞，ILT4仅分布在单核和树突状细胞等髓样细胞；KIR2DL4主要表达在CD56bright NK细胞亚群。
HLA-G分子的免疫调节功能分为两个方面：一方面，HLA-G（主要指HLA-G1、-G5抗原）能直接调节机体免疫细胞的生物学功能，主要表现在：① 有效抑制CTL和NK细胞的免疫杀伤活性；② 诱导CD8+ T细胞或CD8+ NK细胞的凋亡；③ 抑制CD4+ T细胞增殖或阻碍初始型T细胞的分化；④ 抑制树突状细胞等抗原递呈细胞（antigen presenting cell, APC）的成熟和活化，影响抗原递呈；⑤ HLA-G能够刺激免疫效应细胞上的ILT2、ILT4、KIR2DL4等抑制性受体的表达上调等；⑥ HLA-G能刺激APC细胞分泌TGF-β、IL-10等细胞因子，使Th1/Th2平衡移向Th2。另一方面，HLA-G对免疫系统的间接调节，通过诱导产生调节性细胞发挥长效的免疫耐受机制，主要表现在：① 诱导产生耐受型树突状细胞，继而分泌IL-10细胞因子；② 诱导产生CD4+CD25highFoxp3+ Treg细胞；③ 刺激初始型T细胞分化获得CD3+CD4low 和CD3+CD8low 抑制性T细胞亚群；④ 通过“trogocytosis”机制，HLA-Gacq+ T细胞具备HLA-G抗原的免疫学功能等。因此， HLA-G分子是机体内一个重要的免疫耐受分子。
HLA-G1、-G2、-G3、-G4、-G5、-G6、-G7在表达机制及作用模式上存在差异，提示不同HLA-G异构体分子可能在各种病理生理过程中发挥特定的免疫学效应。HLA-G异构体分子量不同，研究不同组织细胞HLA-G异构体分子表达及其表达谱，对于阐述特定HLA-G异构体分子的生物学功能及其临床意义具有重要意义。
目前尚缺乏用于免疫印迹等科研方法基于分子量不同区分HLA-G异构体分子的标准品，需要制备高纯度的特定HLA-G异构体分子作为参照。因此，针对各类肿瘤细胞系HLA-G抗原的差异表达，如何建立特异且能稳定表达HLA-G异构体分子的肿瘤细胞系成为该领域急需解决的重要问题。
发明内容
本发明的目的在于提供一种稳定、特异性表达膜结合型抗原HLA-G1的肿瘤细胞株。
特异性表达HLA-G1抗原的细胞株K562，其保藏编号为CCTCC NO：C201351。命名为表达HLA-G1的K562细胞株，保藏日期为2013年5月8日，保藏单位是中国典型培养物保藏中心，保藏地址是中国武汉武汉大学。
进一步地，所述细胞株表达的HLA-G1核酸序列如SEQ ID NO.3所示，氨基酸序列如SEQ ID NO.4所示。
由该细胞株生产的HLA-G1分子量为39kDa，包括重链、轻链及抗原肽；其中重链包括α1、α2、α3 结构域和跨膜区、胞内肽段，轻链即β2微球蛋白（β2m），分子量12kDa，由人类第15 号染色体编码。该蛋白特异性强，稳定性好，具有很好的商业化应用价值。
本发明HLA-G1肿瘤细胞株的建株方法如下：采用分子生物学方法分离来源于人绒癌细胞株JEG-3的HLA-G1异构体编码基因与真核细胞表达质粒载体pVITRO2-mcs重组，并将其转染至HLA表达阴性的白血病细胞系K562中进行表达，通过多种分子生物学方法鉴定转染细胞系HLA-G1异构体分子的表达情况，完成首轮初筛。初筛后的细胞株，通过有限稀释法进行肿瘤细胞株的克隆化培养，复筛以确定HLA-G1蛋白的表达量高低，挑选表达量最高的细胞株，命名为K562-HLA-G1，-196℃液氮保存，每1-2月复苏一管细胞检验其细胞活力。
采用传统的细胞株鉴定与分子生物学鉴定相结合的方式对筛选得到的细胞株进行鉴定。本发明采用RT-PCR、免疫细胞化学法、Western blot及流式细胞术等技术方法对目的基因及蛋白表达进行鉴定，整个发明过程具有操作简单，设计完整等特点。结果证明表达的HLA-G1异构体分子量完全符合文献报道，本发明建立稳定表达膜结合型HLA-G1抗原的细胞系K562，实现了HLA-G1抗原成功在HLA-G1表达阴性的肿瘤细胞系中的特异性表达。因此，本发明建立的稳定表达的HLA-G1异构体分子可用于各种HLA-G1研究过程中的检测标准品。
在本说明书上下文中，除非特别指明，否则所引用的任何技术术语具有本领域普通技术人员在本领域中通常理解的含义，而未注明的实验方法是按照常规实验方法进行或按照供应商所建议的操作说明进行。
附图说明
图1为HLA-G7种异构体的表达模式图。
图2为K562-HLA-G1构建成功细胞系HLA-G1基因序列测序结果图。
图3 为HLA-G1 异构体的核苷酸和氨基酸序列分析图。
图4为RT-PCR技术鉴定转染细胞中HLA-G1 mRNA表达的电泳图，其中泳道1：核酸Marker；泳道2：K562-HLA-G1细胞。
图5为流式细胞术鉴定转染细胞中HLA-G1的膜蛋白表达的结果。
图6为Western blot技术鉴定转染细胞中HLA-G1异构体蛋白分子量，其中泳道1：K562-pv细胞；泳道2：K562-HLA-G1细胞。
具体实施方式
以下实施例提供了实现本发明的优选实施方案。本领域技术人员可以理解的是，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。下面的实施例中所公开的技术表示由本发明人所发现的技术，这些技术能有效实施本发明。不过，本领域技术人员可以理解，在不超出本发明的构思的前提下，可以对这些具体实施方案进行多种改变，仍然能获得相似的结果。
实施例1：HLA-G1基因克隆和pVITRO2-mcs-HLA-G1重组质粒的构建
分别编码含酶切位点的HLA-G1基因序列的PCR扩增：设计如下一对引物序列：
上游引物，划线部分为EcoR I酶切位点: 5’-TCGAGAATTCATGGTG GTCATGGCGCCCCGAA-3’；(SEQ ID NO.1)
下游引物，划线部分为Xho I酶切位点: 5’-TCGACTCGAGTCAATC TGAGCTCTTCTT -3’ (SEQ ID NO.2)。
以人绒癌细胞株JEG-3的基因组为模板，按照以下条件进行HLA-G1 PCR扩增，片段大小为1037bp。
PCR体系：
H2O: 50μL
Buffer(10×): 5μL
Mg2＋(25mmol/L)： 2μL
Primer-up(25μmol/L): 2μL
Primer-down(25μmol/L): 2μL
dNTP(20mmol/L): 1μL
Template(Genome of FRI 100，10ng/μL)： 1μL
Taq Polymerase(5U/μL): 1μL
PCR程序：
1、95℃预变性5min
2、94℃变性60 s，60℃退火60 s，72℃延伸2min
3、依步骤2循环35次
4、72℃延伸10 min
含编码HLA-G1基因序列的pGEM-T重组质粒的构建：将PCR扩增所得的HLA-G1基因克隆入pGEM-T质粒，转化至大肠杆菌DH5α中扩增。提取该重组质粒，经酶切鉴定后送样测序，结果显示获得的编码HLA-G1的基因序列与http://www.anthonynolan.org.uk网站报道的基因序列相比，与HLA-G*01:01:03序列一致。测序结果如图2所示。通过DNA STAR 软件，膜结合型HLA-G1 异构体的核苷酸和氨基酸序列分析如图3所示，包括信号肽、α1、α2、α3、跨膜区及胞内结构（其中划线部分为信号肽、加框部分为α2、黑体部分是α3、跨膜区及胞内结构为斜体部分所示）。
含编码HLA-G1基因序列的pVITRO2-mcs重组表达质粒的构建：用EcoR I和Xho I分别酶切上述步骤中得到的含内切酶位点的编码HLA-G1基因片段和pVITRO2-mcs质粒。分别回收酶切后的含酶切位点的编码HLA-G1基因片段以及pVITRO2-mcs质粒酶切产物的大片段，并将两者连接，构建了表达质粒pVITRO2-mcs-HLA-G1。将该重组质粒转入大肠杆菌DH5α扩增并提取质粒，经过EcoR I和Xho I酶切鉴定，结果表明目的基因片段已插入载体质粒。目的基因在pVITRO2-mcs-HLA-G1质粒上的详细序列见图3。
实施例2：稳定表达HLA-G1抗原的K562细胞系鉴定
采用RT-PCR鉴定转染细胞中HLA-G1的mRNA表达：Trizol 试剂分别提取各转染细胞株总mRNA 后，经甲醛变性琼脂糖凝胶电泳鉴定未见降解，A260/280 比值为2.0019。取2μl 总mRNA，按Fermentas 公司逆转录酶试剂盒说明操作，合成cDNA第一条链。PCR 反应参数为：94 ℃预变性4 min ;94 ℃ 1 min ,60 ℃ 1 min，72 ℃ 2min，共35 个循环; 最后72 ℃延伸10 min。取5μl PCR 产物进行琼脂糖凝胶电泳，观察结果。RT-PCR扩增所得特异性条带均符合预期目的片段长度：HLA-G1（1037bp）（如图4所示）。结果说明HLA-G1表达阴性的K562细胞成功表达HLA-G1异构体分子。
采用流式细胞术鉴定转染细胞中HLA-G1的蛋白表达：细胞培养至对数生长期，收集细胞，PBS洗涤后，采用MEM-G/9抗体标记后发现：K562-HLA-G1细胞株成功表达膜结合型HLA-G1分子（如图5所示）。
采用Western blot法鉴定转染细胞中HLA-G1蛋白的分子量：取出10μl细胞裂解物行SDS-PAGE,其中JEG-3和K562细胞裂解液分别作为阳性及阴对照。电转膜后,用5%去脂奶粉室温封闭4h,0.2% TBS(Teween-20 PBS) 洗涤。加入HLA-G1 单抗 4H84,4℃孵育过夜,洗涤;加入HRP 标记兔抗鼠IgG抗体, 室温孵育30min,洗涤, 最后，用Dako REAL EnVision 检测系统（DAKO）孵育1-3min。研究结果显示：各转染细胞株内的HLA-G1抗原大小与预期的一致，为39kDa（如图6所示）。
本发明建立的稳定表达的HLA-G1异构体分子可用于各种HLA-G1研究过程中的检测标准品。
SEQUENCE LISTING
<110> 浙江省台州医院
<120> 特异性表达HLA-G1抗原的细胞株K562
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 32
<212> DNA
<213> 人工序列
<400> 1
tcgagaattc atggtggtca tggcgccccg aa 32
<210> 2
<211> 28
<212> DNA
<213> 人工序列
<400> 2
tcgactcgag tcaatctgag ctcttctt 28
<210> 3
<211> 1017
<212> DNA
<213> HLA-G1
<400> 3
atggtggtca tggcgccccg aaccctcttc ctgctactct cgggggccct gaccctgacc 60
gagacctggg cgggctccca ctccatgagg tatttcagcg ccgccgtgtc ccggcccggc 120
cgcggggagc cccgcttcat cgccatgggc tacgtggacg acacgcagtt cgtgcggttc 180
gacagcgact cggcgtgtcc gaggatggag ccgcgggcgc cgtgggtgga gcaggagggg 240
ccagagtatt gggaagagga gacacggaac accaaggccc acgcacagac tgacagaatg 300
aacctgcaga ccctgcgcgg ctactacaac cagagcgagg ccagttctca caccctccag 360
tggatgattg gctgcgacct ggggtccgac ggtcgcctcc tccgcgggta tgaacagtat 420
gcctacgatg gcaaggatta cctcgccctg aacgaggacc tgcgctcctg gaccgcagcg 480
gacactgcgg ctcagatctc caagcgcaag tgtgaggcgg ccaatgtggc tgaacaaagg 540
agagcctacc tggagggcac gtgcgtggag tggctccaca gatacctgga gaacgggaag 600
gagatgctgc agcgcgcgga cccccccaag acacacgtga cccaccaccc tgtctttgac 660
tatgaggcca ccctgaggtg ctgggccctg ggcttctacc ctgcggagat catactgacc 720
tggcagcggg atggggagga ccagacccag gacgtggagc tcgtggagac caggcctgca 780
ggggatggaa ccttccagaa gtgggcagct gtggtggtgc cttctggaga ggagcagaga 840
tacacgtgcc atgtgcagca tgaggggctg ccggagcccc tcatgctgag atggaagcag 900
tcttccctgc ccaccatccc catcatgggt atcgttgctg gcctggttgt ccttgcagct 960
gtagtcactg gagctgcggt cgctgctgtg ctgtggagga agaagagctc agattga 1017
<210> 4
<211> 338
<212> PRT
<213> HLA-G1
<400> 4
Met Val Val Met Ala Pro Arg Thr Leu Phe Leu Leu Leu Ser Gly Ala
1 5 10 15
Leu Thr Leu Thr Glu Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe
20 25 30
Ser Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala
35 40 45
Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ser
50 55 60
Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu Gln Glu Gly
65 70 75 80
Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala His Ala Gln
85 90 95
Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr Asn Gln Ser
100 105 110
Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys Asp Leu Gly
115 120 125
Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala Tyr Asp Gly
130 135 140
Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala
145 150 155 160
Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala Ala Asn Val
165 170 175
Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu
180 185 190
His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg Ala Asp Pro
195 200 205
Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr Glu Ala Thr
210 215 220
Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Ile Leu Thr
225 230 235 240
Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu Leu Val Glu
245 250 255
Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val
260 265 270
Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu
275 280 285
Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Lys Gln Ser Ser Leu Pro
290 295 300
Thr Ile Pro Ile Met Gly Ile Val Ala Gly Leu Val Val Leu Ala Ala
305 310 315 320
Val Val Thr Gly Ala Ala Val Ala Ala Val Leu Trp Arg Lys Lys Ser
325 330 335
Ser Asp
Claims (3)
1.特异性表达HLA-G1抗原的细胞株K562，其保藏编号为CCTCC：C201351。
2.如权利要求1所述菌株的细胞株，其特征在于，所述细胞株表达的HLA-G1核酸序列如SEQ ID NO.3所示。
3.如权利要求1所述菌株的细胞株，其特征在于，所述细胞株表达的HLA-G1氨基酸序列如SEQ ID NO.4所示。
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