CN111363037B - 一种包含特异性结合afp蛋白的抗体的疾病检测试剂盒 - Google Patents
一种包含特异性结合afp蛋白的抗体的疾病检测试剂盒 Download PDFInfo
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- CN111363037B CN111363037B CN202010451891.8A CN202010451891A CN111363037B CN 111363037 B CN111363037 B CN 111363037B CN 202010451891 A CN202010451891 A CN 202010451891A CN 111363037 B CN111363037 B CN 111363037B
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Abstract
本发明涉及一种包含特异性结合AFP蛋白的抗体的疾病检测试剂盒，其包含能够和AFP特异性结合且不与HAS交叉反应的抗体，所述试剂盒拥有更高的灵敏度，用于AFP异常的肝癌、胎儿先天性疾病的早期筛查诊断、疗效检测和预后判断。
Description
技术领域
本发明涉及生物医药领域，具体的涉及一种包含特异性结合AFP蛋白的抗体的疾病检测试剂盒。
背景技术
原发性肝癌是预后极差的恶性肿瘤之一，目前我国肝癌已处于恶性肿瘤病死率的第2位。尽管目前肝癌能够早期发现，且治疗方法较多，但治疗效果仍不能令人满意，大部分肝癌患者对化学药物治疗和放射治疗等均无显著效果，能手术根治的患者仅占15％。甲胎蛋白(alpha—fetoprotein，AFP)是一种重要的肿瘤相关抗原。目前AFP是原发性肝癌诊断和生物治疗的一种重要的靶抗原。AFP是胎儿特异性蛋白，主要在胎儿卵黄囊和肝脏合成，是单链多肽，属于α球蛋白，分子量约为64—72KD，其肽链氨基酸序列与白蛋白相似，其蛋白部分由590个氨基酸组成。血清AFP大于400ng／mL是诊断原发性肝癌的重要辅助指标。研究表明，AFP具有较强的单独促进肝癌细胞增殖作用，并且AFP在肝癌时的高表达，可能是重要的内源性促肿痛细胞增殖的自分泌现象。AFP的检测已成为原发性肝癌诊断和预后监测的重要指标，也成为公认的肝癌生物治疗的靶抗原。除了原发性肝癌，某些胃癌、恶性畸胎瘤、胰腺腺泡细胞癌、结肠癌及肾细胞癌患者均发现血清中AFP水平显著增高。此外，AFP 在胎儿血液循环中浓度较高，出生后 2～3 月 AFP 含量较少，血液中较难检出。若胎儿先天性脊柱裂、神经管缺陷以及唐氏综合征等会导致甲胎蛋白含量的变化，妊娠期可通过 AFP 监测进行上述疾病的筛查和诊断。因此AFP的检测，有助于AFP异常的肝癌、胎儿先天性疾病的诊断与提前干预、治疗。
现有技术中也有一些专利中公开了AFP抗体，例如：CN105037546A、CN10331959B、CN105037544A，但经过发明人研究后发现，这些抗体在灵敏度上还有待提高。同时由于人AFP与人血清白蛋白（Human Serum Albumin，HSA）具有50％同源性，以AFP为抗原免疫后制备的单克隆抗体与人血清白蛋白存在较高的交叉反应几率。
发明内容
基于上述发现，本发明的首要目的在于提供一种新的AFP抗体，具有更高的灵敏度同时不与HSA反应，以解决现有AFP抗体灵敏度低且有可能与HSA发生非特异性结合的问题。
本发明提供以下技术方案：
一种抗AFP抗体，命名为hAFP-2，由重链和轻链组成，重链和轻链分别包括可变区和恒定区，其中重链可变区为SEQ ID NO :1，轻链可变区为SEQ ID NO :2 。
一种抗AFP抗体，命名为hAFP-6，由重链和轻链组成，重链和轻链分别包括可变区和恒定区，其中重链可变区为SEQ ID NO : 9，轻链可变区为SEQ ID NO : 10 。
hAFP-2重轻链可变区共有6个互补决定区。所述抗体1的重链可变区3个CDR序列分别为SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5；轻链可变区3个CDR序列分别为SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8。
所述hAFP-6的重链可变区3个CDR序列分别为SEQ ID NO:11、SEQ ID NO:12、SEQID NO:13；轻链可变区3个CDR序列分别为SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16。
本发明所述AFP抗体的重链和轻链进一步包含恒定区，所述抗体轻链恒定区进一步包含鼠源κ、λ链序列。所述抗体重链恒定区进一步包含鼠源IgG1、IgG2a、IgG2b或IgG3或IgA或IgM序列。
本发明的目的在于提供两种新的AFP抗体，所述抗体均不与HSA交叉反应且识别不同的AFP表位，用于检测样本中AFP蛋白存在及含量的抗体。
本发明的另一目的在于提供一种测定AFP的方法，所述方法使用两种以上不同的AFP抗体，所述的抗体不与HAS交叉反应且识别不同的AFP表位。所述方法为双抗体夹心ELISA检测法。
本发明的又一目的在于提供一种更灵敏的AFP抗体试剂盒，其包含上述识别不同AFP表位的两种抗体，用于AFP异常的肝癌、胎儿先天性疾病等的早期筛查诊断、疗效检测和预后判断。
有益效果
本发明的有益效果主要体现在：所述抗体不与人血清白蛋白交叉反应；所述AFP抗体识别不同AFP表位；由本发明抗体建立的AFP检测试剂盒具有更的灵敏性，为AFP异常的疾病，如肝癌、胎儿先天性疾病的早期诊断提供帮助。
附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。
图1为人AFP双抗体夹心ELISA试剂盒灵敏度检测图。
具体实施方式
以下结合附图对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。
实施例1. 抗AFP抗体制备及纯化
将AFP抗原 (浓度为 1µg/µl)与等体积完全福氏佐剂（CFA）充分混匀形成水状油包后，通过腹腔注射，初次免疫BALB/C 小鼠，每只小鼠200µl。3 周后进行第一次加强，将AFP抗原(浓度为 1µg/µl)与不完全福氏佐剂（IFA）等体积混匀乳化，采用背部多点注射，每只小鼠200µl。以后每隔2周加强免疫一次，每次免疫后第7天取尾血，用 ELISA 法检测抗体效价。当抗体效价达到 10-4左右时，将AFP抗原(浓度为 1µg/µl)与不完全福氏佐剂（IFA）等体积混匀乳化，采用背部多点注射加强免疫一次，每只小鼠200µl。于第四天取脾，进行细胞融合。取免疫后的Balb/c小鼠脾细胞，将其与骨髓瘤Sp2/0细胞株融合，将融合后的细胞加HAT 铺板，3天之后半换液,一周之后全换液,换HAT 培养基培养。12天后，取细胞上清，高通量ELISA法检测上清中与AFP蛋白表现出阳性反应的原代培养物。再将此孔内的杂交瘤细胞进行稀释进行亚克隆，同样以ELISA方法进行筛选，最终筛选出阳性杂交瘤细胞株6株，分别命名为hAFP1-6。将上述杂交瘤细胞株分别扩大培养，收取此杂交瘤细胞，用PBS重悬，配置成107/ml的杂交瘤细胞悬液。用弗氏不完全佐剂作为诱导剂。预先向8周左右的Balb/c腹腔注射弗氏不完全佐剂，3天后，向预先处理过的Balb/c小鼠腹腔中注射500ul浓度为107/ml的杂交瘤细胞悬液。待小鼠腹部肿胀***，采集腹水纯化抗体。经过饱和硫酸铵（PH=7.8）沉淀获得粗提的AFP单抗，再经过protein G柱层析获得纯度较高的AFP抗体。
实施例2.抗体与人血清白蛋白交叉反应实验
AFP蛋白与人血清白蛋白同源性较高，易发生交叉反应，影响AFP抗体的特异性，因此采用交叉反应实验，筛选出不与HAS交叉反应的抗体。将96孔酶标板中加入人血清白蛋白，每孔100ng，4℃孵育过夜。去上清，每孔中加入200μl PBST洗液，静置2min，扣去洗液，重复洗涤3次，每次洗涤尽可能拍干酶标板。加入牛血清白蛋白，37℃封闭1h。去上清，每孔中加入200μl PBST洗液，静置2min，扣去洗液，重复洗涤3次。将实施例1中制备的抗体分别加入检测孔中，37℃孵育2h。去上清，每孔中加入200μl PBST洗液，静置2min，扣去洗液，重复洗涤3次，每次洗涤尽可能拍干酶标板。加入羊抗鼠IgG-HRP标记抗体，37℃孵育1h。去上清，每孔中加入200μl PBST洗液，静置2min，扣去洗液，重复洗涤3次，每次洗涤尽可能拍干酶标板。加入显色底物，反应15min。加入终止液，终止显色反应。将酶标板放入酶标仪中，检测450nm波长处的吸光值。根据各样品OD450的值，计算P/N值。P/N值==(样品OD-空白对照OD)／(阴性对照OD对照-空白对照OD)，若P/N大于2.1为阳性,说明该AFP样品与人血清白蛋白发生交叉反应；若P/N小于2.1为阴性,说明该AFP样品不与人血清白蛋白发生交叉反应，仅特异性结合AFP蛋白。实验结果如表一所示：hAFP-1和hAFP-4为阳性，hAFP-2、hAFP-3、hAFP-5、hAFP-6为阴性。
表一 AFP抗体与人血清白蛋白交叉反应
实施例3. 竞争抑制性ELISA实验
因建立双夹心ELISA检测体系，需要两个结合不同表位的AFP抗体，因此通过竞争抑制ELISA实验，筛选结合不同表位的AFP抗体。将hAFP-6抗体用辣根过氧化物酶（HRP）标记，将其作为酶标的对照抗体。其中HRP标记技术为本领域熟知技术。将hAFP-2、hAFP-3、hAFP-5作为未标记的待检抗体。将AFP 酶标抗体加入到包被 AFP 抗原的96孔酶标板中，实验组则分别加入不同比例的待测抗体（待测抗体：酶标抗体=1，2，4，8，16，32），同时，设立加PBS的AFP 酶标抗体阳性对照；37℃孵育1h；去上清，每孔中加入200μl PBST洗液，静置2min，扣去洗液，重复洗涤3次，每次洗涤尽可能拍干酶标板。加入显色底物，反应15min。加入终止液，终止显色反应。将酶标板放入酶标仪中，检测450nm波长处的吸光值。根据各样品OD450的值，计算抑制率。抑制率（%）=[(OD酶标抗体–OD空白孔)-(OD待测抗体–OD空白孔)]/ （OD酶标抗体–OD空白孔）×100%。最终结果如表二所示：当AFP酶标抗体hAFP-6浓度固定时，其与AFP 抗原混合反应的抑制率随着hAFP-5抗体浓度的增加而逐步增大；且当 hAFP-5：hAFP-6为 32 时，竞争抑制率达到 72.3%，结果表明hAFP-5和hAFP-6结合相同的表位。而hAFP-2、hAFP-3的抑制率在待测抗体：酶标抗体=32 时，仅为8.7%和11.6%。结果表明hAFP-2、hAFP-3与hAFP-6结合不同的表位，均可以与hAFP-6建立双夹心ELISA检测体系。
表二 竞争抑制性ELISA实验
实施例4.双抗体夹心ELISA试剂盒灵敏度检测
经过筛选，最终选用hAFP-2和hAFP-6配对建立双夹心ELISA体系，其中hAFP-2作为包被抗体，hAFP-6-HRP作为检测抗体。将抗AFP抗体hAFP-2用包被液稀释至0.5μg/ml，100μl/孔，4℃过夜孵育，将其固定于酶标板上。去上清，每孔中加入200μl PBST洗液，静置2min，扣去洗液，重复洗涤3次，每次洗涤尽可能拍干酶标板。选用3%脱脂牛奶作为封闭液，每孔中加入100μl，室温封闭1h。去上清，每孔中加入200μl PBST洗液，静置2min，扣去洗液，重复洗涤3次，每次洗涤尽可能拍干酶标板。将AFP抗原（按照600ng/ml，150 ng/ml，37.5ng/ml，9.37ng/ml，2.34ng/ml，0.59ng/ml，0.15ng/ml的梯度进行稀释。将稀释后的抗原依次加入酶标板中，每孔100μl，37℃孵育30min。去上清，每孔中加入200μl PBST洗液，静置2min，扣去洗液，重复洗涤3次，每次洗涤尽可能拍干酶标板。将辣根过氧化物酶（HRP）标记的抗AFP抗体hAFP-6，即hAFP-6-HRP作为检测抗体，将抗体稀释至0.5μg/ml，每孔中加入100μl，室温孵育1h。去上清，每孔中加入200μl PBST洗液，静置2min，扣去洗液，重复洗涤3次，每次洗涤尽可能拍干酶标板。每孔中加入100μlTMB，室温避光孵育15-30min。每孔中加入50μl终止液，终止显色反应。将酶标板放入酶标仪中，检测450nm波长处的吸光值。最终得到的数据为图1所示，最低检测线0.15ng/ml,线性范围为0.59ng/ml-150ng/ml，线性关系为0.986。而根据现有技术例如CN105037544A中双夹心标准曲线结果，其检测到的最低浓度为1.21ng/ml。因此利用本发明所述的AFP抗体制备的双夹心ELISA检测试剂盒，其灵敏度明显高于现有技术CN105037544A中的试剂盒。
实施例5. 单克隆抗体序列确定
细胞制备：将hAFP-2、hAFP-6对应的杂交瘤细胞株复苏后，培养至总数量107 个细胞，1000rpm离心5min收集细胞，提取RNA。向细胞沉淀中加入TRNzol-A+裂解，室温静止15min。每ml TRNzol-A+加入200μl氯仿，漩涡振荡15秒，放置3分钟。13000 rpm 4°C离心10分钟，Trizol-A+ 细胞溶液分为三层：上层无色水相、中层和下层黄色有机相，将溶有RNA的水相转移至离心管中，向水相中加入等体积异丙醇，混匀，室温静置25分钟。13000 rpm 4°C离心10分钟，弃去废液得到沉于底部的RNA沉淀。用75%乙醇洗涤RNA沉淀两次后，用PEDC水溶解RNA，-80℃保存。用反转录试剂盒SMARTerRACE合成第一链cDNA，以第一链cDNA为后续模板扩增杂交瘤细胞所对应的抗体可变区DNA序列。设计特异性的巢式PCR引物，该扩增反应中所使用的引物序列与抗体可变区第一框架区和恒定区互补，采用常规PCR方法扩增目的基因。将扩增产物测序后，得到杂交瘤克隆hAFP-2分泌的抗AFP抗体hAFP-2的重链可变区序列SEQ ID NO：1和轻链可变区序列SEQ ID NO：2；该抗体的重链可变区3个CDR序列分别为SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5；轻链可变区3个CDR序列分别为SEQ ID NO:6、SEQID NO:7、SEQ ID NO:8。杂交瘤克隆hAFP-6分泌的抗AFP抗体hAFP-6的重链可变区序列SEQID NO：9和轻链可变区序列SEQ ID NO：10；该抗体的重链可变区3个CDR序列分别为SEQ IDNO:11、SEQ ID NO:12、SEQ ID NO:13；轻链可变区3个CDR序列分别为SEQ ID NO:14、SEQ IDNO:15、SEQ ID NO:16。
序列表
<110> 北京瀚梅生物科技有限公司
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<400> 2
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Lys Asn Gly Ser Asp Ile Glu
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Glu Gly Thr Leu Lys Leu Leu Ile
35 40 45
Tyr Tyr Ser Gly Ile Glu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gly Thr Asp Ser Tyr Ile Arg
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 3
<211> 5
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
Asp Ala Thr Lys Gln
1 5
<210> 4
<211> 17
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
Thr Ile Gly Asp Gln Tyr Trp Glu Arg Leu Gln Phe Arg Met Ser Lys
1 5 10 15
Gly
<210> 5
<211> 9
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 5
Arg Tyr Gly Lys Ser Asn Gln Ile Tyr
1 5
<210> 6
<211> 11
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
Arg Ala Lys Asn Gly Ser Asp Ile Glu Leu Asn
1 5 10
<210> 7
<211> 7
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 7
Tyr Ser Gly Ile Glu Gln Ser
1 5
<210> 8
<211> 9
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 8
Gln Gly Thr Asp Ser Tyr Ile Arg Thr
1 5
<210> 9
<211> 118
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 9
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Ala
20 25 30
Asp Gly Gln Trp Val Lys Gln Arg Arg Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asp Gly Lys Phe Gly Ser Arg Asn Pro Tyr Ile Gln Trp
50 55 60
Met Gly Arg Ala Thr Leu Thr Arg Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Lys Gly Ala Thr Ile Lys Pro Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210> 10
<211> 107
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 10
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Cys Ser Cys Asp Tyr Asn Lys Gly Ala Thr Ile Lys
20 25 30
Pro Asn Trp Tyr Gln Gln Lys Pro Glu Gly Thr Leu Lys Leu Leu Ile
35 40 45
Tyr Tyr Gly Pro Lys Leu Asn Val Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Asn Gly Thr Asp Glu Ile Arg
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 11
<211> 5
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 11
Arg Ala Asp Gly Gln
1 5
<210> 12
<211> 17
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 12
Leu Ile Asp Gly Lys Phe Gly Ser Arg Asn Pro Tyr Ile Gln Trp Met
1 5 10 15
Gly
<210> 13
<211> 9
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 13
Asn Lys Gly Ala Thr Ile Lys Pro Tyr
1 5
<210> 14
<211> 11
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 14
Asp Tyr Asn Lys Gly Ala Thr Ile Lys Pro Asn
1 5 10
<210> 15
<211> 7
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 15
Tyr Gly Pro Lys Leu Asn Val
1 5
<210> 16
<211> 9
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 16
Gln Asn Gly Thr Asp Glu Ile Arg Thr
1 5
Claims (6)
1.一种AFP蛋白检测试剂盒，其特征在于：该试剂盒是双抗体夹心ELISA试剂盒，包含与AFP特异性结合的两个不同抗体hAFP-2和hAFP-6，所述两个不同抗体分别如下：
a）hAFP-2的重链可变区3个CDR序列分别为SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5；轻链可变区3个CDR序列分别为SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8；
b）hAFP-6的重链可变区3个CDR序列分别为SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13；轻链可变区3个CDR序列分别为SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16。
2.一种AFP蛋白检测试剂盒，其特征在于：该试剂盒是双抗体夹心ELISA试剂盒，包含与AFP特异性结合的两个不同抗体hAFP-2和hAFP-6，所述两个不同抗体分别如下：
a）hAFP-2的重链可变区序列如SEQ ID NO :1所示，和轻链可变区序列如SEQ ID NO :2所示；
b）hAFP-6的重链可变区序列如SEQ ID NO :9所示，和轻链可变区序列如SEQ ID NO :10所示。
3.两个抗体的组合在用于制备检测肝癌的试剂盒中的用途，其特征在于：两个不同抗体hAFP-2和hAFP-6分别如下：
a）hAFP-2的重链可变区序列如SEQ ID NO :1所示，和轻链可变区序列如SEQ ID NO :2所示；
b）hAFP-6的重链可变区序列如SEQ ID NO :9所示，和轻链可变区序列如SEQ ID NO :10所示。
4.两个抗体的组合在用于制备检测AFP蛋白的试剂盒中的用途，其特征在于：两个不同抗体hAFP-2和hAFP-6分别如下：
a）hAFP-2的重链可变区序列如SEQ ID NO :1所示，和轻链可变区序列如SEQ ID NO :2所示；
b）hAFP-6的重链可变区序列如SEQ ID NO :9所示，和轻链可变区序列如SEQ ID NO :10所示。
5.两个抗体的组合在用于制备检测肝癌的试剂盒中的用途，其特征在于：两个不同抗体hAFP-2和hAFP-6分别如下：
a）hAFP-2的重链可变区3个CDR序列分别为SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5；轻链可变区3个CDR序列分别为SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8；
b）hAFP-6的重链可变区3个CDR序列分别为SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13；轻链可变区3个CDR序列分别为SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16。
6.两个抗体的组合在用于制备检测AFP蛋白的试剂盒中的用途，其特征在于：两个不同抗体hAFP-2和hAFP-6分别如下：
a）hAFP-2的重链可变区3个CDR序列分别为SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5；轻链可变区3个CDR序列分别为SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8；
b）hAFP-6的重链可变区3个CDR序列分别为SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13；轻链可变区3个CDR序列分别为SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16。
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