中国旱獭Ⅰ型干扰素受体β亚基克隆、表达及功能初步鉴定

陶颖; 杨东亮; 王宝菊; 刘艺; 桂文甲; 李智; 樊和斌

doi:10.12449/JCH240210

中国旱獭Ⅰ型干扰素受体β亚基克隆、表达及功能初步鉴定

DOI: 10.12449/JCH240210

陶颖¹,
杨东亮²,
王宝菊²,
刘艺¹,
桂文甲¹,
李智¹,
樊和斌^1, , ,

1.
中国人民解放军中部战区总医院感染科，武汉 430030
2.
华中科技大学同济医学院附属协和医院感染科，武汉 430030

基金项目:

国家自然科学基金 (3021170);

国家重大基础研究项目（973） (2005CB522900)

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：陶颖、樊和斌负责课题设计，资料分析，撰写论文；李智、刘艺、桂文甲参与收集数据，修改论文；杨东亮、王宝菊对课题进行指导；樊和斌负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
樊和斌， 296592559@qq.com （ORCID： 0000-0002-9602-7755）

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出版历程
- 收稿日期: 2023-04-11
- 录用日期: 2023-07-04
- 出版日期: 2024-02-19

Preliminary identification of the cloning， expression， and function of Marmota himalayana type I interferon receptor β subunit

1.
Department of Infectious Diseases，General Hospital of The Central Theater Command，Wuhan 430030，China
2.
Department of Infectious Diseases，Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology，Wuhan 430030，China

Research funding:

National Natural Science Foundation of China (3021170);

National Major Basic Research Projects （973） (2005CB522900)

More Information

Corresponding author: FAN Hebin， 296592559@qq.com （ORCID： 0000-0002-9602-7755）

摘要

摘要: 目的克隆中国旱獭Ⅰ型干扰素受体β亚基（mhIFNAR2）的基因，并进行抗体制备及功能鉴定。方法应用RT-PCR技术，从中国旱獭脾组织中扩增得到序列，克隆至原核表达载体pRSET-B，表达重组蛋白，电泳和Western Blot法鉴定；重组蛋白常规免疫BALB/c小鼠制备其胞外段多克隆抗体，免疫组化、免疫荧光和Western Blot法鉴定；再通过siRNA阻断的方法检测其功能。计量资料多组间比较采用方差分析，进一步两两比较采用LSD-t检验。结果从mhIFNAR2扩增出149～1 300 bp片段，其同源性在分析的种属中以土拨鼠最高，可达98.05%。成功地构建了表达胞外段mhIFNAR2_{（50-181aa）}蛋白的原核表达质粒，命名为pRSET-B.mhIFNAR2；其表达重组蛋白分子量27 kD，纯化后纯度约为95%，浓度约为160 μg/mL。用纯化的重组蛋白常规免疫BALB/c小鼠后，获得1∶1 000的特异性多克隆抗体，用免疫组化及免疫荧光可见细胞膜、细胞质有表达。合成的三条siRNA，其中有一条起始于277位点的siRNA（siRNA277）与空白对照及阴性对照相比，可以沉默目的基因的表达，并能减弱干扰素的信号通路（P值均<0.05）。结论获得mhIFNAR2的部分序列，成功地制备出抗mhIFNAR2胞外段多克隆抗体，该抗体有较高的效价和特异性，并能用于免疫组化、免疫荧光及Western Blot的检测。用siRNA277可以抑制目的基因的表达，并能阻断干扰素的信号通路。
- 受体，干扰素αβ /
- 克隆 /
- 乙型肝炎
Abstract: Objective To clone the gene of Marmota himalayana type ‍Ⅰ interferon receptor β subunit （mhIFNAR2）， and to perform antibody preparation and functional identification. Methods RT-PCR was used for amplification in the spleen tissue of Marmota himalayana to obtain the sequence， which was cloned to the prokaryotic expression vector pRSET-B to express the recombinant protein. Electrophoresis and Western blot were used for identification. BALB/c mice were immunized with the recombinant protein to prepare the polyclonal antibody of its extracellular domain； immunohistochemistry， immunofluorescence assay， and Western Blot were used for identification， and the method of siRNA blockade was used to investigate its function. An analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for comparison between two groups. Results A fragment of mhIFNAR2 （149‍ ‍—‍ ‍1 ‍300 bp） was obtained from spleen tissue， which showed the highest homology of 98.05% in marmot. A prokaryotic expression plasmid was successfully constructed for expression of the extracellular domain of the mhIFNAR2_{（50-181aa）} and was named pRSET-B.mhIFNAR2， and the recombinant protein expressed by this plasmid had a molecular weight of 27 kD， a purity of about 95% after purification， and a concentration of 160 μg/mL. After BALB/c mice were immunized with the purified recombinant protein， 1∶1 000 specific polyclonal antibodies were obtained， and immunohistochemistry and immunofluorescence assay showed the expression in cell membrane and cytoplasm. Among the three siRNAs synthesized， the siRNA starting from the 277 locus （siRNA277） could silence the expression of target genes and weaken the interferon signaling pathway compared with the blank control group and the negative control group （both P<0.05）. Conclusion The fragment of mhIFNAR2 is obtained， and the polyclonal antibody for the extracellular domain of mhIFNAR2 is successfully prepared， with relatively high titer and specificity， and can be used for immunohistochemistry， immunofluorescence assay， and Western blot.
- Receptor， Interferon alpha-beta /
- Clone /
- Hepatitis B

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肝细胞癌（HCC）是临床常见的恶性肿瘤之一。HCC早期治疗以手术切除为主，但侵袭性HCC的治疗策略目前选择有限，索拉非尼、仑伐替尼及程序性细胞死亡受体1及其配体免疫抑制剂治疗HCC的效果不佳，临床有效率不足30%。因此，亟需进一步探究HCC的发病机制与治疗靶点。随着基因组学研究的发展，对HCC肿瘤生物学的认识逐渐深入，但HCC的泛素化特征尚不明晰。本研究旨在揭示HCC的泛素化特征，并探寻可指导侵袭性HCC临床诊断和治疗的潜在生物标志物。

陆军军医大学第一附属医院（重庆西南医院）谢传明教授、张雷达教授与重庆医科大学侯宇教授等通过对85例HCC患者肿瘤及相邻正常肝组织进行蛋白质组学、磷酸化修饰组学和泛素化修饰组学测序与分析发现，COL4A1、LAMC1和LAMA4在无病生存期较差患者中高表达。磷酸化与泛素化修饰在代谢和转移相关信号通路中存在关联。利用泛素化组学和蛋白质组学数据对HCC进行分子分型，发现具有不同临床特征的3个亚型：S-‍Ⅰ、S-‍Ⅱ和S-Ⅲ。S-‍Ⅰ亚型以代谢相关蛋白高表达为特征，预后最好；S-Ⅲ亚型与增殖/转移信号通路密切相关，预后最差，表现为总生存期最短和复发率最高；而S-‍Ⅱ亚型则介于两者之间。研究发现，生物标志物TUBA1A、BHMT2、BHMT和ACY1的表达表现出不同的泛素化水平，与HCC患者预后不良密切相关，表明靶向这些蛋白质或其泛素化修饰蛋白可能对临床治疗有益。此外，研究证实TUBA1A K370去泛素化可驱动HCC发生与转移，这主要归因于AKT介导的USP14激活。TUBA1A K370去泛素化标志着一类高侵袭性HCC亚型。值得注意的是，靶向AKT-USP14-TUBA1A复合物可促进TUBA1A降解，并在体内阻断HCC发生。

总之，本研究从泛素化组学、磷酸化组学和蛋白质组学角度对HCC进行了全面的整合分析，该研究不仅加深了对HCC中泛素化特征的了解，并为开发新的HCC生物标志物和潜在的治疗靶点提供了依据。

摘译自LIN XT, LUO YD, MAO C, et al. Integrated ubiquitomics characterization of hepatocellular carcinomas[J]. Hepatology, 2024. DOI: 10.1097/HEP.0000000000001096. [Online ahead of print]

(陆军军医大学第一附属医院肝胆外科谢传明报道）

图 1 mhIFNAR2片段核苷酸组成物种同源性比较

Figure 1. Species homology comparison of mhIFNAR2

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图 2 SDS-PAGE电泳分析His-mhIFNAR2蛋白的表达

Figure 2. Expression of His-mhIFNAR2 analyzed by SDS-PAGE

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图 3 pRSET-B.mhIFNAR2蛋白纯化图

Figure 3. Purification of pRSET-B.mhIFNAR2

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注： L1，中分子量的蛋白质标准；L2，mhIFNAR2多抗血清作为一抗（1∶1 000）；L3，mhIFNAR2多抗血清作为一抗（1∶10 000）；L4，正常小鼠血清作为一抗；L5，兔抗His作为一抗。

图 4 纯化的目的蛋白Western Blot检测

Figure 4. Western Blot analysis of the activity and specificity of antisera

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图 5 mhIFNAR2多克隆抗体的免疫组化结果（DAB，×400）

Figure 5. Immunohistochemical of mhIFNAR2 polyclonal antibodies（DAB，×400）

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图 6 PBMC中mhIFNAR2免疫荧光检测

Figure 6. Stained with mouse anti-mhIFNAR2 antibody by immunofluorescence in PBMC

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图 7 不同浓度的siRNA840对WH12-6细胞mhIFNAR2、M×A基因的抑制作用

Figure 7. The inhibitory effect of siRNA840 at different concentrations on mhIFNAR2 and M×A

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表 1 干扰素受体β亚基的引物序列

Table 1. Primer sequences for interferon receptor β subunit

名称	序列	核苷酸位点
mhIFNAR1-2s	5′-AGTACATTTAGAAGCTGAAG-3'	396～416
mhIFNAR1-2as	5'-CTCTTCAGACCAAAAAGATG-3'	943～963
mhIFNAR2-1s	5'-CCATCTTATCATGGGAATTA-3'	149～169
mhIFNAR2-1as	5'-CTCTCAAAAACACAGAGTT-3'	1 281～1 300

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表 2 mhIFNAR2 siRNA 模板序列

Table 2. Template of mhIFNAR2 siRNA

cDNA	正义链（5′-3′）	反义链（3′-5′）
GAAGTAGCTCTCAGAACTA（mhIFNAR1B_840B）	GAAGUAGCUCUCAGAACUAdTdT	dTdTCUUCAUCGAGAGUCUUGAU
GCTTTACAGACCACATTAA（mhIFNAR2B_277B）	GCUUUACAGACCACAUUAAdTdT	dTdTCGAAAUGUCUGGUGUAAUU
GCTGAAGATAAGGCAATAA（mhIFNAR1B_412B）	GCUGAAGAUAAGGCAAUAAdTdT	dTdTCGACUUCUAUUCCGUUAUU
阴性对照	公司未提供序列

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表 3 EMCV感染滴度测定结果

Table 3. Result of EMCV infection titer determination

病毒稀释	有CPE孔	无CPE孔	累计有CPE孔	累计无CPE孔	有/总和	感染百分率（%）
10⁰	4	0	39	0	39/39	100
10^-1	4	0	35	0	35/35	100
10^-2	4	0	31	0	31/31	100
10^-3	4	0	27	0	27/27	100
10^-4	4	0	23	0	23/23	100
10^-5	4	0	19	0	19/19	100
10^-6	4	0	15	0	15/15	100
10^-7	4	0	11	0	11/11	100
10^-8	4	0	7	0	7/7	100
10^-9	2	2	3	2	3/5	60
10^-10	1	3	1	5	1/6	16.7

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表 4 不同浓度的siRNA277对WH12-6细胞mhIFNAR2、M×A基因的抑制作用

Table 4. The inhibitory effect of siRNA277 at different concentrations on mhIFNAR2 and M×A

组别	mhIFNAR2	M×A
空白对照（n=3）	11.678±0.725	3.087±1.910
阴性对照（n=3）	14.190±3.100	4.150±0.500
20 nmol/L（n=3）	2.538±0.124^1）2）	1.350±0.212^1）2）
注：与空白对照组比较，1）P<0.05；与阴性对照组比较，（2）P<0.05。

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