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病毒性神经坏死病(viral nervous necrosis, VNN)是一种传染病,其病原体为神经坏死病毒(Nervous necrosis virus,NNV),该病可引起鱼类病毒性神经坏死,是我国鱼类养殖中危害极大的病毒性疾病之一,特别是在我国东南沿海地区较常见。据报道,30多种海鱼在苗期和培养过程中,曾受到VNN的影响[1-2],其对石斑鱼仔鱼和幼鱼,会造成很高的死亡率,给海洋鱼类养殖造成较大的经济损失[3-4]。国际兽疫组织(OIE)将该病列为重要的鱼类病害。目前,石斑鱼养殖产业针对该病毒的防疫主要在两方面,一方面是注意在鱼卵、用具、水、饵料生物的消毒。比如用0.2 mg·L−1残存臭氧的海水来清洗鱼卵,场所的再次清洁、干燥及消毒,可提供有效的保护,但是这些措施只能在一定程度上减少感染;另一方面是制备疫苗,对石斑鱼进行免疫接种[5]。对石斑鱼注射病毒性神经坏死病的抗原(MCP)进行免疫接种[6-9]的方法存在成本高、工作量大、不适合对较小幼鱼注射等局限[10],为此该方法不适合在石斑鱼产业中广泛应用。目前通过肌肉内或腹膜内注射的几种类型NNV疫苗已经开发并应用,包括重组衣壳蛋白[11], 病毒样颗粒[12-13]和灭活病毒体[14-15],但注射免疫的方式并不能有效激起鱼的黏膜免疫,且免疫过程费时、费力、费用昂贵。因此,针对目前注射免疫接种方法的局限,笔者将神经坏死病毒的衣壳蛋白(MCP)和鮰爱德华氏菌的外膜蛋白N1(ompN1)进行融合表达,拟利用融合抗原MCP-ompN1制备粘膜疫苗,该粘膜免疫疫苗可通过口服和浸泡的方式使石斑鱼的幼鱼达到免疫和更早保护的目的,同时还可以大批量免疫从而降低免疫成本。
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OmpN1基因序列(GenBank: NC_012779,protein ID: WP_015870209.1)和MCP基因序列(GenBank: AF534998.3)。MCP连接在ompN1的N端,中间用柔性氨基酸链GGGS连接,该重组基因序列MCP-ompN1经海南大学生命科学与药学院生物技术与分子药理实验室优化后,由生工生物工程(上海)股份有限公司合成。
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大肠杆菌(E. coli)DH5α 感受态细胞、BL21(DE3)感受态细胞均购自生工生物工程(上海)股份有限公司;PET28a原核表达载体和pMD19-T(sample)克隆载体均由海南大学生命科学与药学院生物技术与分子药理实验室保存;胶回收试剂盒、质粒小提试剂盒均购自TaKaRa公司;T4 DNA连接酶购自Promega公司;BamH I和Sal I限制性内切酶均购自New England Biolabs公司;透析袋购自Solarbio公司(货号:YA1072);氢氧化铝凝胶为本实验室自制;不完全佐剂和完全佐剂购自sigma公司。
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BALB/c小鼠雌性,6~8周龄,体质量约20 g,购自广东省医学实验动物中心。
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将优化合成的MCP-ompN1融合基因利用BamH I和Sal I双酶切后回收,连接至经BamH I和Sal I双酶切回收的PET-28a载体上,构建MCP-ompN1 pET-28a原核表达重组质粒。
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根据已经进行密码子优化后的MCP基因序列以及pET-28a表达载体上的BamH I和Sal I酶切位点,设计1对含有酶切位点的特异性引物。上游引物为F1:5′-CGCGGATCCATGGTTCGTAAAGGTGAAAAAAAAC-3′;下游引物为R1:5′-ACGCGTCGACTTATTAGTTTTCAGAGTCAACACGGGTGCAA-3′;其中引物F1下划线表示的是BamH I酶切位点,引物F2下划线表示的是Sal I酶切位点。以MCP-ompN1重组基因序列作为模板,扩增出目的基因MCP,其片段大小和预期的一致为1 017 bp。将扩增并回收的目的基因MCP连接到pMD19-T(simple) 克隆载体中,连接产物转化至感受态细胞DH5α后,依次通过菌落PCR鉴定,质粒PCR鉴定,双酶切鉴定和最后的测序确定MCP pMD19-T重组质粒构建成功。重组克隆质粒MCP pMD19-T(simple) 和表达质粒pET28a进行BamH I和Sal I的双酶切,再通过胶回收试剂盒从MCP pMD19-T(simple) 酶切产物中得到目的基因MCP,与BamH I和Sal I双酶切后pET28a连接,最终完成原核表达质粒MCP pET28a的构建。
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同样根据已经进行密码子优化后的鮰爱德华氏菌外膜蛋白OmpN1基因序列,设计含有酶切位点的上下游引物来扩增OmpN1的基因序列,上游酶切位点为BamH I,引物的序列为F2:5′-CGCGGATCCGCTGAAATCTACAACAAAAACG-3′;下游酶切位点为Sal I,引物的序列为R2:5′-ACGCGTCGACTTATTAGAAGTTGTACTGG -3′。其中,限制性内切酶位点用下划线表示。以MCP-ompN1重组基因序列作为模板,扩增出目的基因OmpN1,其片段大小和预期的一致为1 146 bp。将扩增并回收的目的基因OmpN1连接到pMD19-T(simple)克隆载体中,连接产物转化至感受态细胞DH5α后,同样通过菌落PCR鉴定,质粒PCR鉴定,双酶切鉴定和最后的测序确定ompN1 pMD19-T重组质粒构建成功。
重组克隆质粒ompN1 pMD19-T(simple) 和表达质粒pET28a均进行BamH I和Sal I双酶切,将酶切产物OmpN1片段通过胶回收试剂盒进行回收,与双酶切后的表达载体pET28a连接,完成原核表达质粒ompN1 pET-28a的构建。
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将原核表达质粒MCP-ompN1 pET28a,MCP pET28a,ompN1 pET28a,分别转入大肠杆菌BL21(DE3)感受态细胞中,挑取单菌落至10 mL含有卡那霉素的LB培养基中,37 ℃ 培养箱中振摇过夜。次日,按体积比为1∶100转接培养至OD600≈0.6。取出1 mL菌液作为非诱导对照。然后将剩下的菌液分别取1 mL至EP管中,每个蛋白共8管。设置不同IPTG浓度诱导实验。IPTG的浓度设置为0.1,0.2,0.3,0.4,0.5,0.6,0.8,1.0 mmol·L−1,37 ℃ 诱导4 h,收集等量菌体,SDS-PAGE蛋白电泳检测,确定每个蛋白所需的最适合IPTG诱导浓度。
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将每个重组蛋白用其最适的IPTG诱导浓度进行诱导表达,对诱导表达后的菌液进行离心并收集菌体, 反复冻融后进行超声波破碎,离心后用上清及沉淀进行SDS-PAGE电泳鉴定。其中,沉淀用含2 mol·L−1尿素和2 mol·L−1盐酸胍的洗涤液洗涤,洗涤后8 000 r·min−1,10 min离心弃上清收集沉淀,连续洗涤3次,然后用含8 mol·L−1尿素缓冲液溶解沉淀,至沉淀完全溶解为止。将溶解液8 000 r·min−1离心10 min,弃沉淀取上清。
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MCP-ompN1/ompN1/MCP蛋白均在包涵体中表达,所以要进行包涵体的复性及纯化,具体的步骤如下。
(1)包涵体的纯化大量诱导表达的菌液以6 000~8 000 r·min−1,4 ℃,离心15 min,弃上清;用9倍体积的菌体破碎缓冲液I振荡重悬菌体,并用超声破碎仪在冰浴条件下超声破碎。超声破碎条件:功率40 %,工作5 s,暂停3 s,破碎15 min,8 000 r·min−1(50 mL离心管转速,以下同)离心15 min。将离心收集的包涵体依次用PBS缓冲液、缓冲液II、缓冲液III进行包涵体沉淀的洗涤,最终放置10 min。4 ℃,8 000 r·min−1离心15 min收集沉淀。然后按照每克初始湿菌体加100 µL包涵体溶解缓冲液I,重悬包涵体,缓慢振摇1 h。4 ℃,8 000 r·min−1离心15 min,收集上清。
(2)蛋白质透析将蛋白浓度调整至0.1~1.0 g·L−1,装入透析袋中,放置于复性缓冲液I进行梯度透析复性。梯度透析复性步骤:1)在含6 mol·L−1脲素的复性缓冲液I中,4 ℃ 缓慢透析6 h;2)在含4 mol·L−1脲素的复性缓冲液I中,4 ℃ 缓慢透析6 h;3)在含2 mol·L−1脲素的复性缓冲液I中,4 ℃ 缓慢透析6 h;4)将已经依次进行6,4,2 mol·L−1脲素梯度透析复性的蛋白溶液置于磷酸盐(PBS)缓冲液中透析过夜,然后SDS-PAGE检测蛋白纯度。复性后的蛋白进行镍(Ni)柱纯化,得出复性及纯化后的蛋白MCP-ompN1,MCP及ompN1,最后用超滤管进行蛋白浓缩得到最适浓度,对蛋白进行−80 ℃保存。
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取BALB/c小鼠9只,其中3只注射PBS作为对照。3只注射纯化后ompN1蛋白制备成的抗原,另3只注射纯化后MCP蛋白制备成的抗原。注射前从眼角取0.5 mL血液制备血清,留作对照。免疫方式采用皮下多点注射方式进行,免疫剂量为1 g·L−1抗原;在首次免疫7 d后进行第2次免疫,以后每隔7 d免疫1次,共免疫4次。每次免疫前一天对小鼠进行眼角采血,将采到的血样4 ℃下放置过夜,在血块收缩后,4 ℃下1 200 r·min−1离心10 min分离出血清部分,进行后续的检测。
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将纯化后的MCP-mpN1/ompN1/MCP蛋白进行SDS-PAGE电泳后将胶条割至合适大小,用转膜缓冲液平衡。预先裁好与胶条同样大小的滤纸和NC膜,浸入转膜缓冲液中10 min。转膜装置从下至上依次按阳极碳板、24层滤纸、NC膜、凝胶、24层滤纸、阴极碳板的顺序放好,滤纸、凝胶、NC膜精确对齐,每一步去除气泡,用玻璃棒来回滚动,将碳板上多余的液体吸干。接通电源,恒压25 V,转移1.5 h。转移结束后,断开电源将膜取出,割取待测膜条做免疫印迹。用1×TBST洗膜,5 min×3次,加入封闭液(1x Blocking buffer(表1)加入5% 脱脂奶粉),平稳摇动,室温2 h。弃封闭液,用1× TBST洗膜,15 min×5次。孵血清一抗(按体积比1∶2 000封闭液稀释,液体必须覆盖膜的全部),4 ℃下放置过夜。弃一抗,用1×TBST洗膜,15 min×5次,加入辣根过氧化物酶偶联的二抗(按体积比1∶5 000用封闭液稀释),避光放置2 h。弃二抗,用1×TBST洗膜,15 min×5次。最后通过Western-blot进行分析。
表 1 缓冲液的配方
Table 1. Formulae of buffers
缓冲液 Buffer 配方 Formula 菌体破碎缓冲液I 50 mmol·L−1 Tris-HCl,1 mmol·L−1 EDTA,100 mmol·L−1 NaCl,1% TritonX-100,pH8.5 PBS缓冲液 8.0 g NaCl,28.65 g Na2HPO4·12H2O,0.234 g NaH2PO4·2H2O(1 L)pH8.5 缓冲液II 50 mmol·L−1 Tris-HCl,1 mmol·L−1 EDTA,100 mmol·L−1 NaCl,1% TritonX-100,2 mol·L−1脲素,pH8.5) 缓冲液III 50 mmol·L−1 Tris-HCl,1 mmol·L−1 EDTA,100 mmol·L−1 NaCl,1% TritonX-100,2 mol·L−1 盐酸胍,pH8.5 包涵体溶解缓冲液I 50 mmol·L−1 Tris-HCl,1 mmol·L−1 EDTA,100 mmol·L−1 NaCl,10 mmol·L−1 DTT,2 mmol·L−1脱氧胆酸钠,8 mol·L−1脲素,pH8.5 复性缓冲液I (50 mmol·L−1 Tris-HCI,100 mmol·L−1 NaCl,6 mol·L−1/4 mol·L−1/2 mol·L−1 脲素,1% 甘氨酸,5% 甘油,0.2% PEG(相对分子量3 550),1 mmol·L−1氧化型谷胱甘肽,1mmol·L−1还原型谷胱甘肽,pH8.5 Blocking buffer 10 mmol·L−1 Tris-HCl, pH 7.4, 104 mmol·L−1 NaCl; 25 mmol·L−1 NaF, 8 mmol·L−1 NaN3, 0.1% Tween 20
Prokaryotic Expression of Fusion Gene of Fish Nervous Necrosis Virus Capsid Protein MCP and Edwardsiella Ictaluri Outer Membrane Porin Protein OmpN1
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摘要: 目前在针对鱼类神经坏死病毒的疫苗研究中,主要是将神经坏死病毒某些蛋白作为抗原进行注射免疫,但是传统的注射免疫并不能有效地激发黏膜免疫。笔者将鱼类神经坏死病毒的衣壳蛋白(MCP)与鮰爱德华氏菌的跨粘膜蛋白ompN1融合表达,拟制备能够抵抗神经坏死病毒的粘膜疫苗;利用从NCBI GenBank库里获得的鱼类神经坏死病毒的外壳蛋白MCP和鮰爱德华氏菌的外膜蛋白ompN1的基因序列,将两者进行序列优化与全基因合成,分别构建原核表达载体:MCP-ompN1 pET28a和MCP pET28a和ompN1 pET28a,并在大肠杆菌内分别诱导表达融合蛋白MCP-ompN1,MCP,ompN1后,再利用包涵体纯化及透析复性获得MCP-ompN1,MCP,ompN1蛋白。SDS-PAGE结果显示,原核表达纯化得到了较纯的MCP-ompN1 融合蛋白,Western Blotting结果表明,纯化得到的MCP-ompN1 融合蛋白不仅具有MCP抗原性,还具有ompN1抗原性。本实验通过原核表达纯化得到了鱼类神经坏死病毒衣壳蛋白MCP和鮰爱德华氏菌外膜蛋白ompN1的融合蛋白MCP-ompN1,为进一步验证融合蛋白MCP-ompN1能否作为抵抗神经坏死病毒的粘膜疫苗奠定了基础。Abstract: At present, certain proteins of the nervous necrosis virus are injected into fish as vaccines, but traditional injection immunization cannot effectively stimulate mucosal immunity. In this experiment, the capsid protein (MCP) of the fish nervous necrosis virus was fused with the transmucosal protein ompN1 of Edwardsiella ictaluri to prepare a mucosal vaccine against the nervous necrosis virus. The gene sequences of the coat protein MCP of the fish nervous necrosis virus and the outer membrane protein ompN1 of E. ictaluri were obtained from the NCBI GenBank, and then optimized and synthesized, and the synthesized genes were used to construct prokaryotic expression vectors respectively: MCP-ompN1 pET28a, MCP pET28a, ompN1 pET28a. The fusion proteins MCP-ompN1, MCP and ompN1 were induced and expressed in Escherichia coli, respectively, and they were purified through inclusion body purification and refolded through dialysis. SDS-PAGE results showed that the purified MCP-ompN1 fusion protein was obtained by prokaryotic expression and purification. Further Western blotting showed that the purified MCP-ompN1 fusion protein had both MCP antigenicity and ompN1 antigenicity. It is indicated that the MCP-ompN1 protein into which the fish nervous necrosis virus capsid protein MCP was fused with E. ictaluri outer membrane protein ompN1 was obtained by prokaryotic expression and purification. Further study is needed to observe whether the fusion protein MCP-ompN1 can be used as a mucosal vaccine against nervous necrosis virus.
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Key words:
- viral nervous necrosis /
- Edwardsiella ictaluri /
- fusion protein /
- inclusion body
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图 2 ompN1 pET-28a重组质粒图谱及酶切验证
A. ompN1 pET-28a重组质粒图谱;B. ompN1 pET-28a质粒由BamH I和Sal I双酶切鉴定;M: DL 5 000 bp DNA marker;1: 环状空质粒PET28a对照;2: ompN1阳性对照;3: ompN1 pET-28a重组质粒由BamH I和Sal I双酶切后得到线性化空质粒PET28a和目的片段ompN1。
Fig. 2 Map of ompN1 pET-28a recombinant plasmid and enzyme digestion verification
A. The ompN1 pET-28a recombinant plasmid map; B. The ompN1 pET-28a plasmid was identified by double digestion with BamH I and Sal I; M: DNA marker DL 5 000; 1: Circular empty plasmid PET28a control; 2: ompN1 positive control; 3: The ompN1 pET-28a recombinant plasmid was double digested with BamH I and Sal I to produce a linearized empty plasmid PET28a and a target fragment ompN1.
图 3 MCP pET-28重组质粒图谱及酶切验证
A. MCP pET-28重组质粒图谱;B. MCP pET-28重组质粒由BamHI和SalI双酶切鉴定;M: DL 5 000 bp DNA marker;1: 环状空质粒PET28a对照;2: MCP阳性对照;3: MCP pET-28a重组质粒被BamH I和Sal I双酶切后得到线性化空质粒PET28a和目的片段MCP。
Fig. 3 MCP PET28a recombinant plasmid map and enzyme digestion verification
A. MCP pET-28 recombinant plasmid map; B. MCP pET-28 recombinant plasmid was identified by BamH I and Sal I double digestion; M: DNA marker DL 5 000; 1: Circular empty plasmid PET28a control; 2: MCP positive control; 3: The MCP pET-28a recombinant plasmid was digested with BamH I and Sal I to produce a linearized empty plasmid PET28a and a target fragment MCP.
图 4 MCP-ompN1 PET28a重组表达载体的梯度诱导
M: 120 kDa Protein Marker;1:未加IPTG对照组;2-9: 分别为0.1,0.2,0.3,0.4,0.5,0.6,0.8,1.0 mmol·L−1的IPTG浓度。
Fig. 4 MCP-ompN1 PET28a recombinant expression vector gradient induction
M: 120 kDa Protein Marker; 1: Control group without IPTG; 2-9: IPTG at the concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 mmol·L−1, respectively.
图 5 ompN1 pET-28a表达载体的梯度诱导
M: 120 kDa Protein Marker;1: 未加IPTG对照组;2-9: 分别为0.1,0.2,0.3,0.4,0.5,0.6,0.8,1.0 mmol·L−1的IPTG浓度。
Fig. 5 Gradient induction of ompN1 pET-28a expression vector
M: 120 kDa Protein Marker; 1: Control group without IPTG; 2-9: IPTG at the concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 mmol·L−1, respectively.
图 6 MCP pET-28a表达载体的梯度诱导
M: 120 kDa Protein Marker;1: 未加IPTG对照组;2~9: 分别为0.1,0.2,0.3,0.4,0.5,0.6,0.8,1.0 mmol·L−1的IPTG浓度。
Fig. 6 MCP pET-28a expression vector gradient induction
M: 120 kDa Protein Marker; 1: control group without IPTG; 2−9: IPTG at the concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 mmol·L−1, respectively.
图 7 融合蛋白的可溶性表达检测
M: 120 kDa Protein Marker;A. MCP-ompN1;1: 未加IPTG诱导;2: 加入0.1 mmol·L−1 IPTG的全菌诱导;3: 诱导表达后破碎上清;4: 诱导表达后破碎沉淀。B. ompN1;1: 加入0.2 mmol·L−1 IPTG的全菌诱导;2: 诱导表达后破碎上清;3: 诱导表达后破碎沉淀。C. MCP;1: 加入0.2 mmol·L−1 IPTG的全菌诱导;2: 诱导表达后破碎上清;3: 诱导表达后破碎沉淀。
Fig. 7 Soluble expression detection of the fusion protein
M: 120 kDa Protein Marker; A. MCP-ompN1; 1: Induction without IPTG; 2: Whole bacteria induction with addition of 0.1 mmol·L−1 IPTG; 3: Supernatant after fragmentation after induction; 4: Fragmentation after induction of expression precipitation. B. OmpN1; 1: Whole bacteria induction with addition of 0.2 mmol·L−1 IPTG; 2: Supernatant broken after induction of expression; 3: Broken pellet after induction of expression. C. MCP; 1: Whole bacteria induction with addition of 0.2 mmol·L−1 IPTG; 2: Broken supernatant after induction of expression; 3: Broken precipitate after induction of expression.
图 8 SDS-PAGE检测MCP-ompN1/MCP/ompN1蛋白
M: 120 kDa Protein Marker;A. MCP-ompN1;1:未加IPTG诱导;2: 加入0.1 mmol·L−1IPTG的全菌诱导;3: 复性及纯化后MCP-ompN1融合蛋白。B. MCP;1: 未加IPTG诱导;2: 加入0.2 mmol·L−1 IPTG的全菌诱导;3: 复性及纯化后MCP蛋白。C.ompN1;1: 未加IPTG诱导;2: 加入0.2 mmol·L−1 IPTG的全菌诱导;3: 复性及纯化后ompN1蛋白。
Fig. 8 SDS-PAGE detection of MCP-ompN1/ MCP/ ompN1 protein
M: 120 kDa Protein Marker; A. MCP-ompN1; 1: Induction without IPTG; 2: Whole bacteria induction with addition of 0.1 mmol·L−1 IPTG; 3: Renaturation and purification of MCP-ompN1 fusion protein. B. MCP; 1: Induction without IPTG; 2: Whole bacteria induction with 0.2 mmol·L−1 IPTG; 3: Renaturation and purified MCP protein. C. OmpN1; 1: Induction without IPTG; 2: Whole bacteria induction with addition of 0.2 mmol·L−1 IPTG; 3: Renaturation and purification of ompN1 protein.
图 9 纯化后的融合蛋白MCP-ompN1的Western blot检测
M: 120 kDa Protein Marker;A中1: ompN1蛋白;2: 融合蛋白MCP-OmpN1;B中1: MCP蛋白;2: 融合蛋白MCP-OmpN1。
Fig. 9 Western blotting of purified fusion protein MCP-ompN1
M: 120 kDa Protein Marker; A. 1: OmpN1 protein; 2: Fusion protein MCP-OmpN1; B. 1: MCP protein; 2: Fusion protein MCP-OmpN1.
表 1 缓冲液的配方
Table 1 Formulae of buffers
缓冲液 Buffer 配方 Formula 菌体破碎缓冲液I 50 mmol·L−1 Tris-HCl,1 mmol·L−1 EDTA,100 mmol·L−1 NaCl,1% TritonX-100,pH8.5 PBS缓冲液 8.0 g NaCl,28.65 g Na2HPO4·12H2O,0.234 g NaH2PO4·2H2O(1 L)pH8.5 缓冲液II 50 mmol·L−1 Tris-HCl,1 mmol·L−1 EDTA,100 mmol·L−1 NaCl,1% TritonX-100,2 mol·L−1脲素,pH8.5) 缓冲液III 50 mmol·L−1 Tris-HCl,1 mmol·L−1 EDTA,100 mmol·L−1 NaCl,1% TritonX-100,2 mol·L−1 盐酸胍,pH8.5 包涵体溶解缓冲液I 50 mmol·L−1 Tris-HCl,1 mmol·L−1 EDTA,100 mmol·L−1 NaCl,10 mmol·L−1 DTT,2 mmol·L−1脱氧胆酸钠,8 mol·L−1脲素,pH8.5 复性缓冲液I (50 mmol·L−1 Tris-HCI,100 mmol·L−1 NaCl,6 mol·L−1/4 mol·L−1/2 mol·L−1 脲素,1% 甘氨酸,5% 甘油,0.2% PEG(相对分子量3 550),1 mmol·L−1氧化型谷胱甘肽,1mmol·L−1还原型谷胱甘肽,pH8.5 Blocking buffer 10 mmol·L−1 Tris-HCl, pH 7.4, 104 mmol·L−1 NaCl; 25 mmol·L−1 NaF, 8 mmol·L−1 NaN3, 0.1% Tween 20 -
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