CN103923934B - 一种具有免疫负调控作用的基因工程蛋白及其制备方法与应用 - Google Patents
一种具有免疫负调控作用的基因工程蛋白及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于分子免疫学技术领域，具体为一种具有免疫负调控作用的基因工程蛋白CPL及其制备方法与应用。本发明首先提供一种CPL基因，其核苷酸序列如SEQ ID NO：1所示。本发明还提供上述基因所编码的融合蛋白CPL，其氨基酸序列SEQ ID NO：2所示。本发明还提供前述融合蛋白CPL的制备方法和免疫调控效果（免疫抑制）以及用途。本发明提供的基因工程蛋白可以有效阻止T细胞活化，具有显著免疫抑制作用，可用于制备免疫抑制剂。本发明为研发防治自身免疫疾病，抗移植排斥及超敏反应的药物开辟广阔前景。
Description
技术领域
本发明属于分子免疫学技术领域，具体涉及一种具有免疫负调控作用的基因工程蛋白CPL及其制备方法与应用。
背景技术
免疫反应过度激活引起的疾病在临床上非常普遍，包括类风湿性关节炎，红斑狼疮等自身免疫病和器官移植急性排斥等。对机体非正常的免疫应答的调控研究一直以来都是免疫学主要任务之一。免疫应答过激的主要机制在于体内病理性淋巴细胞的激活，使T细胞攻击自体组织或移植组织，同时产生抗体，造成移植物排斥或炎症反应。早期临床上主要应用糖皮质激素和钙调磷酸酶抑制剂等副作用较大的免疫抑制药物进行治疗
传统的免疫抑制药物如糖皮质激素，烷化剂和抗代谢药物，如临床常用的FK506和Ciclosporin A等虽然有效，但特异性仍不够理想。开发更安全，特异性更好的免疫抑制剂一直是免疫学的重要研究方向。新的药物要求能在抑制对异体器官排斥的同时，并不降低病原菌和肿瘤的正常免疫应答。由于在一个完整免疫应答的过程中，T细胞的激活需经历:T细胞表面的T细胞受体与抗原递呈细胞表面的MHC多肽复合物结合的第一信号；抗原递呈细胞表面的B7与T细胞表面的CD28分子结合的共刺激信号；除此之外，T细胞活化增殖分化还需要IL－2等细胞因子的帮助，称为第三信号。因此新一代的免疫抑制剂可特异性作用于淋巴细胞活化的这几个信号传导通路中，阻断T细胞活化信号，诱导免疫无反应性或无能。
本发明所涉及蛋白，具有两个功能结构域，能在有效抑制共刺激信号的同时，传导免疫负调控信号，特异性的高效抑制T细胞活化。
发明内容
本发明目的在于提供一种具有高效免疫负调控作用的基因工程蛋白CPL及其制备方法与应用。
本发明提供的具有高效免疫负调控作用的新型基因工程蛋白，其成分具有两个功能结构域，分别为结构域P和结构域C，并在两个功能结构域之间引入了柔性铰链区，以使两个结构域能分别正确折叠，记为CPL。该基因工程蛋白也称为免疫负调控分子，
本发明首先提供一种基因，该基因的核苷酸序列如SEQ ID NO: 1所示，命名为CPL基因。
本发明还提供一种重组载体，它包含前述CPL基因。其中，所述的重组载体是重组pet28a质粒。
本发明还提供一种基因工程重组的可表达CPL蛋白质的原核和真核细胞（菌株及细胞株），即重组菌,它包含所述的重组基因及其重组载体。所述重组菌可以为大肠埃希氏杆菌。
本发明还提供前述基因编码的蛋白质，命名为CPL分子（即融合蛋白或基因工程蛋白）。其中，所述蛋白质的氨基酸序列如SEQ ID NO: 2所示。
本发明还提供前述融合蛋白的制备方法。
I.取所述的重组菌，以1:10~1:100的接种量转接于LB培养基中；200rpm/min培养至OD600为0.2～1.0之间；
II．加入终浓度为0.1~1.0mmol的IPTG为诱导剂，温度为30~40℃，诱导培养2~24h；
III．收集菌体，破壁，分离纯化，得到目的蛋白。
上述方法中，优选参数为：所述的重组菌以1:25-1：50的接种量转接于LB培养基中，培养至OD600为0.4～0.6之间；诱导剂终浓度为：0.2~0.5mmol，诱导温度30~40℃，诱导时间2~6h。
研究表明，上述融合蛋白CPL分子，具有高效免疫负调控作用，可以用于制备一种高效免疫抑制剂。具体地说，该融合蛋白可抑制CD28与B7分子结合，进而抑制T细胞活化。另外，该融合蛋白通过与CD279结合产生免疫负调控信号，进而起到免疫抑制作用。
本发明还提供一种新型免疫抑制剂，其有效成分为前述蛋白质。
附图说明
图1 结构域P和结构域C以及新型基因工程蛋白全长编码基因的PCR。
图2 新型基因工程蛋白原核表达载体阳性转化子的PCR鉴定。
图3 SDS-PAGE分析新型基因工程蛋白Ni-NTA亲和柱纯化产物。
图4 Western Blot鉴定新型基因工程融合蛋白。
图5 混合淋巴反应结果。
图6 ConA转化实验结果。
具体实施方式
合成如下引物用于克隆：
1）结构域P第一外显子克隆引物
DomainP-U1： 5’-TCTCTCGAGAAAAGATTATTCACAGTGACAGT-3’ （SEQ ID NO: 3）
XhoI
DomainP -D1: 5’-TAAGGTCTCACTTTGACTTTCAGAGT-3’ （SEQ ID NO: 4）
Eco31I
2）结构域P第二外显子+第三外显子克隆引物
DomainP-U2: 5’-TATGGTCTCAAAAGCTTCCTACAGGAAAATAAA-3’ （SEQ ID NO: 5）
Ecol31I
DomainP-D2: 5’-ATAGCGGCCGCTTAATGATGATGATGATGATGAGTTGGATGGGT
NotI
CCTGGGTTCCATCTGACTTTGAAGGTCAATGC-3’ （SEQ ID NO: 6）
3）全长蛋白克隆引物
Comp-U1 ：5’-AGCCATATGTTATTCACAGTGACAG-3’ （SEQ ID NO: 7）
NdeI
Comp-D1：5’-ATAGGATCCACCGCCAGTTGGATGGGTCCTGGGTT-3’（SEQ ID NO: 8）
BamHI
Comp-U2：5’-ATAGGATCCATGCATGTTGCTCAACC-3’ （SEQ ID NO: 9）
BamHI
Comp-D2：5’-ATAGCGGCCGCTTAATCAGAATCTGGAC-3’ （SEQ ID NO: 10）
NotI
1．目的片段的获得
1.1 结构域P外显子的PCR
第一外显子PCR，按如下体系加样
PCR条件：
95℃ 5min → 94℃ 10s → 55℃ 10s → 72℃ 30s,30循环→72℃ 5min。
第二外显子+第三外显子29bp PCR，按如下体系加样
PCR条件95℃ 5min → 94℃ 10s → 55℃ 10s → 72℃ 60s， 30循环→ 72℃5min，PCR产物电泳后，进行胶回收，胶回收步骤：
1) 取5μl PCR产物走1%的琼脂糖电泳，鉴定条带大小无误；
2) 将所有PCR产物上样走1%的琼脂糖电泳；
3) 在紫外灯下将扩增条带切下，放入1.5ml离心管，并称重；
4) 按每100 mg琼脂糖加入100μl DE-A液的比例加入 DE-A液，置70 ℃水浴使琼脂糖完全溶化；
5) 按每100μl DE-A加入300μl DE-B溶液的比例加入DE-B ；
6) 混匀后将液体移入吸附柱，12000rpm离心30s.倒掉收集管中的液体，再将吸附柱放入同一个收集管中；
7) 在吸附柱中加入500μl W1液，12000rpm离心30s.倒掉收集管中的液体，将吸附柱放入同一个收集管；
8) 在吸附柱中加入700μl W2液，12000rpm离心30s.倒掉收集管中的液体，将吸附柱放入同一个收集管；
9) 离心2钟后，将吸附柱放入一个干净的1.5ml的离心管中，在吸附膜中央加入40μl温育过的MilliQ H2O，静置2分钟后，12000rpm离心一分钟。
结构域P外显子的拼接
在第一外显子下游引物和第二外显子上游引物中引入ⅡS型限制性内切酶位点Ecol31I，用同序异尾酶方法实现外显子之间的拼接。
载体及PCR片段的酶切
第一外显子的的酶切：按如下体系加样，37 ℃水浴反应3h
第二+三外显子的酶切：按如下体系加样，37 ℃水浴反应5h
载体双酶切：按如下体系加样，37 ℃水浴反应5h
酶切产物胶回收步骤同前述。
酶切产物连接连接
连接条件：16℃连接过夜。
转化DH5a和转化子鉴定：
试剂配制
1) TSB (5ml): LB3.9ml, 10% PEG3350 0.5ml, 5%DMSO 25μl, 10mM MgCl20.0102g, 10mM MgSO4 0.0123g, 10% 甘油
2) 5×KCM (5ml): 0.5M KCl 0.186g, 0.15M CaCl2 0.083g, 0.25M MgCl20.25g。
感受态细胞的制备
1) 接DH5a单菌落到5mL LB培养液，37℃振摇培养过夜；
2) 取200μl过夜培养物,转接到20mL LB中,于37℃振摇培养约2小时左右，至菌液的OD值达到约0.4；
3) 将菌液4℃，6000rpm离心5min，弃上清；
4) 加入1/10体积的预冷TSB悬浮沉淀,混匀后冰浴10min；
5) 以50μl/管分装于1.5ml离心管中，-80℃保存。
连接产物转化DH5a
1) 取连接产物5μl，MilliQ H2O 35μl，5×KCM 10μl混匀置于冰上；
2) 取出-80℃保存的DH5a感受态细胞冰浴溶化后加入步骤1中的混合物；
3) 冰浴20min，室温放置10min；
4) 加入500μl预热的LB培养基，150rpm振荡45min；
5) 离心吸弃部分培养基后取50μl左右涂布含Kna的LB平板；
6) 平板37 ℃倒置培养过夜。
法鉴定重组子
1) 挑取转化板的菌落，接入0.5ml LB，37 ℃ 220rpm培养5h ；
2) 用上下游引物和Taq酶以2）中所述的离心上清为模板进行PCR扩增检测，体系如下：
20μlPCR鉴定反应体系：
阳性对照加入PCR模版pPIC9K-Domain C 0.5μl，阴性对照不加入任何模版。
3) PCR扩增反应条件：
4) 取10μl PCR产物，1%的琼脂糖电泳检测。
全长蛋白表达载体的构建
利用pPICZa-Domain C和pPICZa-Domain P质粒为模版克隆构建全长CPL蛋白pet28a表达质粒。为了使两个结构域能够正确折叠，在两个分子之间引入柔性铰链区结构，同时由于铰链区编码基因内存在一个BamHI位点，便于两段基因片段的拼接。
DomainP片段的PCR和酶切纯化
DomainP片段PCR，按如下体系加样
PCR条件：
酶切体系如下：
37℃水浴，酶切5h。
DomainC片段的PCR和酶切纯化
DomainC片段PCR，按如下体系加样，PCR条件同前所述
Domain C片段酶切体系如下：
胶回收步骤参照第一章7.1.4和7.1.5所述。
全长CPL基因表达蛋白载体pET28a的构建
pET28a载体用NdeI，NotI双酶切后，用Axygen胶回收试剂盒进行纯化
将pET28a载体和全长编码基因进行连接，连接体系如下：
反应条件：16℃连接过夜；
转化E.coli BL21菌株、PCR鉴定和测序参照1.2.3所述。
IPTG诱导新型重组蛋白表达
1)挑取若干经鉴定阳性克隆子及空载体pET-28a的E.coli BL21转化克隆分别接种于含5ml LB培养基中(含Amp 100μg/ml)，32℃培养过夜；
2)次日以1:50的接种量转接于LB培养基中(含Amp 100μg/ml)，200rpm/min培养至OD600为0.4～0.6之间；
3)加入终浓度为0.3mmol的IPTG为诱导剂,37℃继续培养4h。
非变性条件下融合蛋白的 Ni-NTA agarose亲和纯化
1.5.1全长重组蛋白的大量诱导表达
1) 挑取经测序鉴定正确的E.coli BL21转化菌株接种于含50ml LB培养基中，37℃培养过夜；
2) 次日转接于2L的LB培养基中，200rpm/min培养至OD600为0.4～0.6之间，加入0.3mM的IPTG，30℃诱导培养4h；
3) 6000rpm 5min收集菌体，洗涤后用200ml裂解缓冲液（50mM Tris-HCl, 100mMNaCl, 1mM PMSF, pH8.0）悬浮，1500psi压力破壁；
4) 10000rpm离心收集沉淀，用Ni-NTA柱Binding Buffer重新溶解（20mM Tris，0.5M NaCl，20mM咪唑， PH8.0）；
5) 离心收集上清准备过柱纯化。
Ni-NTA亲和层析柱分离纯化全长重组蛋白
试剂配制
1) Binding Buffer：20mM Tris，0.5M NaCl， 20mM咪唑 PH8.0 ；
2) Elution Buffer：20mM Tris，0.5M NaCl， 500mM咪唑PH8.0 ；
3) MilliQ H2O ；
4) 20%乙醇：量取200ml 无水乙醇，配平至1000ml，上述溶液均用0.45µm滤膜抽滤，以防止柱子堵塞。
纯化方法
1) 用5柱体积的MilliQ H2O洗Ni-NTA agarose预装柱(5ml)至基线平稳，流速5ml/min；
2) 用10个柱体积Binding Buffer预平衡柱子，至基线再次平稳；
3) 将步骤1所得发酵液上柱，流速3ml/min ；
4) 上样结束后用Binding Buffer进行杂蛋白冲洗，流速5ml/min，直至流出液离子强度及UV达到基线位置（即上样前平衡液强度）；
5) 用Elution Buffer洗脱目的蛋白，根据紫外280吸收峰强度收集洗下来的目的蛋白；
6) 用10倍体积的MilliQ H2O洗柱；
7) 用5倍体积的20%乙醇洗柱，4℃保存。
重组蛋白除盐
1) 准备好MilliQ H2O，20%乙醇，用0.45um微孔滤膜过滤 ；
2) 用10柱体积的MilliQ H2O洗Desalting预装柱(5ml)至基线平稳；
3) 将纯化并超滤浓缩好的蛋白上样0.5ml；
4) 用10柱体积的MilliQ H2O洗脱，按OD280峰收集。
凝血酶切除His-tag：
使用凝血酶试剂盒对已经纯化好的重组蛋白进行酶切，按试剂盒要求按照下表加样：
| 10×凝血酶酶切Buffer | 100μl |
| 凝血酶（1U/μl） | 1μl |
| 重组蛋白（2mg/ml） | 500μl |
| MilliQ H2O | 400μl |
酶切条件：21℃ 16h
次日收集酶切完毕的重组蛋白用10KD超滤管超滤去除小分子量的标签，再经去内毒素试剂盒去除内毒素后用于细胞实验。
重组蛋白的混合淋巴细胞反应以及ConA转化实验
1.7.1 1小鼠淋巴细胞分离
1) 分别取BALB/c和C57小鼠各一只，断颈处死后浸泡于75%乙醇中消毒后无菌操作取出脾脏；
2) 加入5ml EZ-Seq Mouse淋巴细胞分离液研磨，转移到离心管中加入500ul1640培养基；
3) 800g离心后吸出淋巴细胞层，再加入10ml的1640培养基颠倒洗涤；
4) 250g离心收集细胞并计数；
5) 用1640培养基调整细胞浓度为1×106，接入96孔培养板中，每孔100ul 。
双向混合淋巴细胞反应
1) 按下表分组，每孔总体积200μl。每组3个复孔
| 组别 | 加样 |
| 阳性刺激组 | B＋C |
| 阴性对照组 | C＋C 或B+B |
| 低剂量实验组 | B+C+2ng/μl 重组蛋白 |
| 高剂量实验组 | B+C+10ng/μl 重组蛋白 |
注:B代表BALB/c小鼠淋巴细胞，C代表C57小鼠淋巴细胞
2) 37度，5％CO2培养箱内培养72h；
3) 每孔加入10ul的CCK8，37℃，5％CO2培养箱继续培养4h；
4) 取出，用M5酶标仪测量OD450；
5) 计算刺激指数SI：（阳性刺激组—实验组）/ 阳性刺激组。
转化实验
方法可参考双向混合淋巴细胞反应，按下表分组：
| 组别 | 加样 |
| 阴性对照组 | B |
| ConA刺激组 | B+5 ng/μl ConA |
| 低剂量实验组 | B + 5 ng/μl ConA + 2ng/μl重组蛋白 |
| 高剂量实验组 | B + 5 ng/μl ConA + 10ng/μl重组蛋白 |
1.7.4数据处理：
刺激指数SI＝反应组CPM 均值/阴性对照组CPM均值。
抑制率（Inhibited rate）＝（1－实验组SI/ ConA刺激组SI）×100％ 。
结果图5和图6显示重组的CPL蛋白在作用浓度2 ng/μl和10 ng/μl时，对双向混合淋巴细胞活化的抑制率分别为45％±2.4和71%±5.0，对ConA转化实验的淋巴细胞活化抑制率分别为52±3.5%和71%±3.0%。
综上，本发明成功的制备了重组蛋白，该融合蛋白可竞争性抑制CD28与B7分子的结合，并通过CD279及其配体途径抑制淋巴细胞的活化，实验显示该融合蛋白具有明显的免疫抑制作用，具有良好的应用前景。
<110> 复旦大学
<120> 一种具有免疫负调控作用的基因工程蛋白及其制备方法与应用
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atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atgttattca cagtgacagt ccctaaggaa ctgtacataa tagagcatgg cagcaatgtg 120
accctggaat gcaactttga cactggaagt catgtgaacc ttggagcaat aacagccagt 180
ttgcaaaagg tggaaaatga tacatcccca caccgtgaaa gagccacttt gctggaggag 240
cagctgcccc tagggaaggc ctcgttccac atacctcaag tccaagtgag ggacgaagga 300
cagtaccaat gcataatcat ctatggggtc gcctgggact acaagtacct gactctgaaa 360
gtcaaagctt cctacaggaa aataaacact cacatcctaa aggttccaga aacagatgag 420
gtagagctca cctgccaggc tacaggttat cctctggcag aagtatcctg gccaaacgtc 480
agcgttcctg ccaacaccag ccactccagg acccctgaag gcctctacca ggtcaccagt 540
gttctgcgcc taaagccacc ccctggcaga aacttcagct gtgtgttctg gaatactcac 600
gtgagggaac ttactttggc cagcattgac cttcaaagtc agatggaacc caggacccat 660
ccaactggcg gtggatccat gcatgttgct caaccagctg ttgttttggc ttcttctaga 720
ggtattgctt cttttgtttg tgaatacgct tctccaggta aggctactga agttagagtt 780
actgttttga gacaagctga ttctcaagtt actgaagttt gtgctgctac ttacatgatg 840
ggtaacgaat tgactttttt ggatgattct atttgtactg gtacttcttc tggtaaccaa 900
gttaacttga ctattcaagg tttgagagct atggatactg gtttgtacat ttgtaaggtt 960
gaattgatgt acccaccacc atactacttg ggtattggta acggtgctca aatttacgtt 1020
attgatccag aaccatgtcc agattctgat taa 1053
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Claims (8)
1.一种基因，称为CPL基因，其特征在于核苷酸序列为SEQ ID NO: 1所示。
2.一种基因工程重组表达载体，其特征在于：该重组表达载体包含权利要求1所述的基因。
3.一种基因工程表达蛋白质，称为CPL蛋白或CPL分子，其特征在于：由权利要求1所述基因编码，其氨基酸序列如SEQ ID NO: 2 所示。
4.一种基因工程重组的可表达CPL蛋白质的原核或真核细胞，即重组菌，其特征在于包含所述的重组基因及其相关的载体。
5.一种如权利要求3所述蛋白质的制备方法，其特征在于具体步骤如下：
I.取权利要求4所述的重组菌，以1:10~1:100的接种量转接于LB培养基中，200rpm/min培养至OD600为0.2～1.0之间；
II．加入终浓度为0.1~1.0mmol的IPTG为诱导剂，温度为30~40℃，诱导培养2~24h；
III．收集菌体，破壁，根据分子量、PI和亲和特性分离纯化并得到目的蛋白。
6.根据权利要求5所述的方法，其特征在于：所述重组菌以1:25-1：50的接种量转接于LB培养基中，培养至OD600为0.4～0.6之间；加入的诱导剂的终浓度为：0.2~0.5mmol，诱导温度30~40℃，诱导时间2~6h。
7.如权利要求4所述的蛋白质在制备免疫抑制剂中的应用。
8.一种免疫抑制剂，其特征在于：有效成分为权利要求3所述的蛋白质。
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