抗利巴韦林单链抗体基因构建及其结构分析

路少鹏; 岳敏; 张欣欣; 李丹; 王文魁; 齐永华

doi:10.19303/j.issn.1008-0384.2021.08.006

抗利巴韦林单链抗体基因构建及其结构分析

doi: 10.19303/j.issn.1008-0384.2021.08.006

路少鹏^{1, 2,},
岳敏²,
张欣欣²,
李丹²,
王文魁^1, ,,
齐永华^2, ,

1.
山西农业大学动物医学学院，山西　太谷　030801
2.
新乡学院动物疫病分子诊断河南省工程实验室，河南　新乡　453000

基金项目: 国家重点研发计划项目（2018YFC1602900）

详细信息

作者简介:
路少鹏（1995−），男，硕士研究生，主要从事药物代谢与药效研究（E-mail：1309909741@qq.com）

通讯作者:
王文魁（1962−），男，博士，教授，主要从事畜禽细胞因子研究（E-mail：wenkui2009@yeah.net）

齐永华（1977−），男，博士，教授，主要从事细菌耐药性及新药开发研究（E-mail：qyh@xxu.edu.cn）

中图分类号: S 859.1
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- 被引次数: 0
出版历程
- 收稿日期: 2021-03-10
- 修回日期: 2021-07-12
- 网络出版日期: 2021-08-10
- 刊出日期: 2021-08-28

Construction and Structure of Anti-ribavirin Single-chain Antibody Gene

LU Shaopeng^{1, 2
,},
YUE Min²,
ZHANG Xinxin²,
LI Dan²,
WANG Wenkui^{1
, ,},
QI Yonghua^{2
, ,}

1.
College of Animal Medicine, Shanxi Agricultural University, Taigu, Shanxi　030801, China
2.
Henan Engineering Laboratory for Molecular Diagnosis of Animal Diseases, Xinxiang University, Xinxiang, Henan　453000, China

摘要

摘要: 目的克隆构建利巴韦林（RBV）单链抗体（scFv）基因，对其理化特性进行分析并对蛋白质结构进行模拟，为后期检测方法的建立及分子改造提供参考依据。方法以分泌利巴韦林抗体的杂交瘤细胞株总RNA为模板，通过RT-PCR技术扩增抗体的重链可变区（VH）和轻链可变区（VL），然后以柔性连接短肽（Gly₄Ser）₃为接头拼接完整的scFv-RBV。利用生物信息学方法对scFv-RBV的理化性质及蛋白结构功能进行预测分析。结果构建的scFv基因编码240个氨基酸，相对分子质量为26 162.27 Da，理论等电点（pI）为8.57。在二级结构中，β-折叠（39.17%）和无规卷曲（45.41%）占主导地位，α-螺旋（5.42%）和β-转角（10%）相对较少。在三级结构中，VH和VL区域被Linker相互牵拉靠近，形成典型的口袋样形状的空间构象，符合单链抗体的结构特征，理论上可以与RBV抗原特异性结合。结论成功构建scFv-RBV基因，并利用生物信息学方法预测分析scFv的二级、三级结构，该结果可为后期进行单链抗体的表达、纯化及定向进化提供理论支撑。
- 利巴韦林（RBV） /
- 单链抗体（scFv） /
- 生物信息学 /
- 结构预测
Abstract: Objective The ribavirin (RBV) single-chain antibody (scFv) gene was constructed, cloned, and physiochemically analyzed to establish a model for the detection method development and molecular modification. Method Using the total RNA of the hybridoma cell line secreting RBV antibody as a template, both heavy-chain (VH) and light-chain variable (VL) regions of the antibody were amplified by RT-PCR. Then the short peptide （Gly₄Ser）₃ was employed as the splicing joint to construct the complete scFv-RBV. Bioinformatics methods were applied to predict and analyze the physiochemical properties, protein structure, and functions of the gene. Results The constructed scFv-RBV encoded 240 amino acids with a relative molecular mass of 26,162.27 Da and a theoretical isoelectric point of 8.57. The secondary structure of the protein consisted of 39.17% β-sheets, 45.41% random coils, 5.42% α-helices, and 10% β-turns. In the tertiary structure, the VH and VL regions were pulled close by Linker, forming a typical pocket-like spatial configuration that conforms to the structural characteristics of a single-chain antibody. Theoretically, it could bind specifically to RBV antigens. Conclusion The successfully constructed scFv-RBV in this study afforded the utilization of bioinformatics methods to predict and analyze the secondary and tertiary structures of scFv gene paving the way for further studies on the expression, purification, and directed evolution of the single-chain antibodies.
- ribavirin（RBV） /
- single chain antibody fragment（scFv） /
- bioinformatics /
- protein structure

HTML全文

图 1 总RNA琼脂糖凝胶电泳

注：泳道1～2：RNA。

Figure 1. Agarose gel electrophoresis of total RNA

Note: Swim lane 1-2: RNA.

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图 2 VH和VL基因的PCR扩增结果

注：M：DL 2 000 DNA marker；泳道1：扩增的VH基因；泳道2：扩增的VL基因。

Figure 2. PCR amplifications of VH and VL genes

Note: M: DL 2 000 DNA marker; Swim lane 1: Amplified VH gene; Swim lane 2: Amplified VL gene.

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图 3 scFv基因的琼脂糖凝胶电泳结果

注：M：DL2 000 DNA marker；泳道1～2：拼接的scFv基因。

Figure 3. Agarose gel electrophoresis of scFv gene

Note: M: DL 2 000 DNA marker; Swim lane 1-2: Spliced scFv gene.

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图 4 重组质粒pCANTAB-5E-scFv的菌液PCR鉴定结果

注：M：DL2 000 DNA marker；泳道1～3：pCANTAB-5E-scFv菌液PCR鉴定结果。

Figure 4. PCR identification on recombinant plasmid pCANTAB-5E-scFv bacterial fluid

Note: M: DL 2 000 DNA marker; Swim lane 1-3: PCR identification results on PCANTAB-5E-scFv bacteria liquid.

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图 5 scFv-RBV氨基酸序列分析结果

Figure 5. Amino acid sequence of scFv-RBV

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图 6 scFv-RBV的二级结构预测

Figure 6. Predicted secondary structure of scFv-RBV

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图 7 scFv-RBV蛋白质三级结构同源建模

Figure 7. Homologous modeling of tertiary structure of scFv-RBV

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表 1 扩增scFv-RBV基因的引物序列

Table 1. Primer sequence for amplifying scFv-RBV

引物名称 Primer name	引物序列（5′-3′） Primer sequence (5'-3')
VH-F	AGG TSM ARC TGC AGS AGT TWGG
VH-B	TGA GGA GAC GGT GAC TGT GGT TCC TTG GCC CC
VL-F	GAC ATT GAG CTC ACC CAG TCT CCC
VL-B	ACG TTT GAT TTC CAG CTT GGT GCC
VH-linker-For	TTTTGGCCCAGCCGGCCCGGAGGTGAAGCTGGTGGA
VH-linker-Back	TCCACCGCCAGAACCTCCGCCACCAGAACCTCCACCGCC TGAGGAGACTGTGAGAGT
VL-linker-For	GGTGGCGGAGGTTCTGGCGGTGGAGGTTCG GA TA TCCAGA TGACACAGT
VL-linker-Back	TTTT GCGGCCGCTTA GGA TACAGTTGGTGCAG
注：VH-linker-For中的划线处为Sfi Ⅰ酶切位点；VL-linker-Back划线处为Not Ⅰ酶切位点； VH-linker-Back和VL-linker-For划线处为Linker序列。 Note: The underline in VH-linker-For is the Sfi Ⅰ restriction site; the underline in VL-linker-Back is the Not Ⅰ restriction site;The underlined VH-linker-Back and VL-linker-For are the Linker sequence.

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表 2 反转录cDNA的反应体系

Table 2. Reaction system of cDNA’s reverse transcription

试剂 Reagent	体积 Volume/μL
5 × PrimeScript RT master mix	4
Total RNA	10
ddH₂O	6
注：反应条件：37 ℃ 15 min；85 ℃ 5 s；4 ℃保存。 Note：Eaction conditions: 37 ℃ for 15 min; 85 ℃ for 5 s; Storage at 4 ℃.

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表 3 PCR扩增VH和VL基因

Table 3. Amplification of VH and VL genes by PCR

样品 Sample	体积 Volume/µL
2 × Taq master mix	10
cDNA	1
VH-F/VL-F	1
VH-R/VL-R	1
ddH₂O	7
注：反应条件：96 ℃ 5 min；96 ℃ 30 s，55 ℃ 30 s，30个循环；72 ℃ 10 min；4 ℃保存。 Note: Reaction conditions: 96 ℃ for 5 min; 96 ℃ for 30 s, 55 ℃ for 30 s, 30 cycles; 72 ℃ for 10 min; Storage at 4 ℃.

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表 4 scFv基因PCR反应体系（step1）

Table 4. PCR reaction system for scFv gene (step 1)

样品 Sample		体积 Volume/µL
2 × phanta max buffer	2 × phanta max buffer	25
dNTP mixture	dNTP mixture	1
HS DNA polymerase	HS DNA polymerase	1
VH	VL	1
VH-linker-For	VL-linker-For	1
VH-linker-Back	VL-linker-Back	1
ddH₂O	ddH₂O	20
注：PCR扩增程序：96 ℃ 90 s；96 ℃ 30 s，63 ℃ 30 s，72 ℃ 30 s，10个循环；72 ℃ 10 min；4 ℃保存。 Note: PCR amplification program: 96 ℃ 90 s; 96 ℃ 30 s, 63 ℃ 30 s, 72 ℃ 30 s, 10 cycles; 72 ℃ 10 min; store at 4 ℃.

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表 5 scFv基因PCR反应体系（step 2）

Table 5. PCR reaction system for scFv gene (step 2)

样品 Sample	体积 Volume/µL
2 × phanta max buffer	25
dNTP	1
HS DNA polymerase	1
上述反应产物 Among all above	10
VH-linker-For	1
VL-linker-Back	1
ddH₂O	11
注：PCR扩增程序同表4。 Note: The PCR amplification procedure is the same as table 4.

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表 6 scFv基因与pCANTAB-5E载体连接体系

Table 6. scFv gene linked with pCANTAB-5E vector

样品Sample	体积Volume/µL
T₄ Ligation buffer（10 ×）	2
T₄ DNA连接酶 T4 DNA ligase	1
scFv酶切纯化产物 scFv enzyme digestion purified product	1
pCANTAB-5E酶切纯化产物 pCANTAB-5E enzyme digestion purified product	3
ddH₂O	13
注：连接条件：16 ℃过夜。 Note: Connection conditions: 16 ℃ overnight.

下载: 导出CSV

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