BLOC1S1基因过表达山羊的生产及生物安全评估

doi:10.11843/j.issn.0366-6964.2025.09.017

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (9): 4328-4340.doi: 10.11843/j.issn.0366-6964.2025.09.017

BLOC1S1基因过表达山羊的生产及生物安全评估

王聪亮¹, 万仕成¹, 陈文博¹, 李剑南¹, 宋岩峰², 杜晓敏², 刘王叶³, 李荣荣³, 雷安民¹, 屈雷², 朱海鲸², 华进联¹

1. 西北农林科技大学动物医学院陕西省干细胞工程技术研究中心, 杨凌 712100;
2. 榆林学院陕西省绒山羊工程技术研究中心, 榆林 719000;
3. 榆林现代农业科技示范区管理委员会, 榆林 719000

收稿日期:2025-02-18 发布日期:2025-09-30
通讯作者: 朱海鲸，主要从事动物繁殖与生殖干细胞研究，E-mail:haijingzhu@yulinu.edu.cn；华进联，主要从事动物干细胞与生殖细胞发育分化调控研究，E-mail:jinlianhua@nwsuaf.edu.cn
作者简介:王聪亮(1996-)，男，陕西榆林人，博士生，主要从事山羊抗病育种研究，E-mail:wcl1996@nwafu.edu.cn；万仕成(1998-)，男，四川凉山人，博士生，主要从事干细胞抗病育种研究，E-mail:shicheng_wan@nwafu.edu.cn。
基金资助:
国家自然科学基金(32072806；32372970)；国家重点研发计划(2022YFD1302201)；陕西省畜牧科技示范和畜牧专项项目(20221086；20230978)；陕西省教育厅重大项目(22JY075)；陕西省农业攻关核心技术(2024NYGG005-6)；西北农林科技大学横向课题(2022HX144；H2023060246)

Production and Biosafety Assessment of BLOC1S1 Gene Overexpression Goats

WANG Congliang¹, WAN Shicheng¹, CHEN Wenbo¹, LI Jiannan¹, SONG Yanfeng², DU Xiaomin², LIU Wangye³, LI Rongrong³, LEI Anmin¹, QU Lei², ZHU Haijing², HUA Jinlian¹

1. Shaanxi Stem Cell Engineering and Technology Research Center, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China;
2. Shaanxi Province Engineering & Technology Research Center of Cashmere Goats, Yulin University, Yulin 719000, China;
3. Management Committee of Yulin Modern Agricultural Science and Technology Demonstration Zone, Yulin 719000, China

Received:2025-02-18 Published:2025-09-30

摘要/Abstract

摘要： 旨在制备BLOC1S1基因过表达山羊，并探究其生物安全性，为抗布鲁氏菌病模型建立和转基因动物遗传育种提供研究模型与平台。本研究以5只经产、健康雌性陕北白绒山羊(2~3岁)为供体进行超数排卵，公羊本交后经输卵管冲胚获得原核胚，通过显微注射系统将BLOC1S1慢病毒浓缩液注射至原核胚透明带间隙后继续培养待其发育至2细胞，再移植至15只经产陕北白绒山羊受体(2~3岁)输卵管内；产羔后，利用PCR、实时荧光定量PCR和Western blotting技术对F0代羔羊进行BLOC1S1基因过表达山羊鉴定，对BLOC1S1基因过表达与野生型山羊的血液生理生化、体重和体高等生长性状指标监测比较。结果显示，原核胚胎显微注射后移植共获得13只羔羊，经PCR检测鉴定，7只为阳性BLOC1S1基因过表达山羊，阳性率为53.8%；qRT-PCR结果显示，BLOC1S1基因在大脑(P<0.01)、脾脏(P<0.001)和肺脏(P<0.05)组织mRNA表达量均显著上调，脾脏组织蛋白水平表达量显著上调(P<0.001)；与野生型山羊相比，BLOC1S1基因过表达山羊WBC、Neu等血液生理指标和TBIL、GLU等血液生化指标均无显著差异(P>0.05)；生长发育速度监测分析结果显示，BLOC1S1基因过表达与野生型山羊初生重、1~6月龄体重和3~6月龄生长性状指标相比无显著差异(P>0.05)。本研究成功生产了BLOC1S1基因过表达山羊，且基因过表达山羊的生长发育、血液生理生化指标与野生型山羊相比无显著差异，进一步为抗布鲁氏菌病模型建立和生物安全评价体系提供理论依据。

关键词: BLOC1S1, 过表达, 山羊, 布鲁氏菌, 生物安全

Abstract: This study aimed to prepared goats that overexpress the BLOC1S1 gene and investigate their biosafety, thereby providing the research model and platform for establishing brucellosis resistance models and for the genetic breeding of transgenic animals. In this study, 5 multiparous and healthy female Shaanbei White Cashmere goats (aged 2 to 3 years) were used as donors for superovulation, and after natural mating with rams, primordial embryos were obtained via fallopian tube flushing, using a microinjection system, BLOC1S1 lentiviral concentrate was injected into the zona pellucida gap of the primordial embryos, which were then cultured until they reached the 2 cell stage before being transferred into the fallopian tubes of 15 multiparous female Shaanbei White Cashmere goat recipients aged 2 to 3 years. After lambing, techniques such as PCR, quantitative real-time reverse tranion PCR, and Western blotting were employed to identify the overexpression of the BLOC1S1 gene in F0 generation lambs, and the physiological and biochemical parameters of blood, along with growth and development indicators, were monitored and compared between BLOC1S1 gene overexpressing goats and wild type goats. The results demonstrated that a total of 13 lambs were obtained following pronuclear microinjection and embryo transfer, and PCR analysis identified 7 positive BLOC1S1 gene overexpressing goats, resulting in a positive rate of 53.8%, furthermore, qRT-PCR results revealed a significant upregulation of BLOC1S1 gene mRNA expression in brain (P<0.01), spleen (P<0.001), and lung (P<0.05) tissues, and additionally, BLOC1S1 protein expression levels were markedly increased in spleen tissue (P<0.001). Compared to wild type goats, goats overexpressing the BLOC1S1 gene exhibited no significant differences in blood physiological parameters, including white blood cell (WBC) count and neutrophil (Neu) levels, nor in biochemical parameters such as total bilirubin (TBIL) and glucose (GLU) (P>0.05). Additionally, the results of growth and development monitoring analyses indicated that there were no significant differences in birth weight, body weight at 1 to 6 months age, or growth traits from 3 to 6 months age between BLOC1S1 gene overexpressing goats and wild type goats (P>0.05). This study successfully prepared goats that overexpress the BLOC1S1 gene, and the growth and development, along with blood physiological and biochemical indicators of these gene overexpressing goats, exhibited no significant differences when compared to wild type goats, which finding provides a theoretical basis for establishing a Brucella disease resistance model and a biosafety evaluation system.

Key words: BLOC1S1, overexpression, goat, brucella, biosafety

中图分类号:

S827.2

王聪亮, 万仕成, 陈文博, 李剑南, 宋岩峰, 杜晓敏, 刘王叶, 李荣荣, 雷安民, 屈雷, 朱海鲸, 华进联. BLOC1S1基因过表达山羊的生产及生物安全评估[J]. 畜牧兽医学报, 2025, 56(9): 4328-4340.

WANG Congliang, WAN Shicheng, CHEN Wenbo, LI Jiannan, SONG Yanfeng, DU Xiaomin, LIU Wangye, LI Rongrong, LEI Anmin, QU Lei, ZHU Haijing, HUA Jinlian. Production and Biosafety Assessment of BLOC1S1 Gene Overexpression Goats[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4328-4340.

参考文献

[1] DAUGALIYEVA A, DAUGALIYEVA S, ABUTALIP A, et al. Study of epidemiological and molecular characteristics of Brucella strains circulating in Kazakhstan[J]. Vet Res Commun, 2025, 49(3): 156.
[2] CAO X, LIU P, WU J, et al. Genome phylogenetic analysis of Brucella melitensis in Northwest China[J]. BMC Microbiol, 2025, 25(1): 208.
[3] FERJANI A, BUIJZE H, KOPPRIO G, et al. A genomic characterization of clinical brucella melitensis isolates from tunisia: integration into the global population structure[J]. Microorganisms, 2025, 13(2): 243.
[4] KALDS P, ZHOU S, CAI B, et al. Sheep and goat genome engineering: from random transgenesis to the CRISPR era[J]. Front Genet, 2019, 10: 750.
[5] LOIS C, HONG E J, PEASE S, et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors[J]. Science, 2002, 295(5556): 868-872.
[6] HOFMANN A, KESSLER B, EWERLING S, et al. Efficient transgenesis in farm animals by lentiviral vectors[J]. EMBO Rep, 2003, 4(11): 1054-1060.
[7] HOFMANN A, ZAKHARTCHENKO V, WEPPERT M, et al. Generation of transgenic cattle by lentiviral gene transfer into oocytes[J]. Biol Reprod, 2004, 71(2): 405-409.
[8] YANG S H, CHENG P H, BANTA H, et al. Towards a transgenic model of huntington’s disease in a non-human primate[J]. Nature, 2008, 453(7197): 921-924.
[9] GÓMEZ M C, POPE C E, KUTNER R H, et al. Generation of domestic transgenic cloned kittens using lentivirus vectors[J]. Cloning Stem Cells, 2009, 11(1): 167-176.
[10] CRISPO M, VILARIÑO M, DOS SANTOS N P C, et al. Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis[J]. Transgenic Res, 2015, 24(1): 31-41.
[11] DENG S, WU Q, YU K, et al. Changes in the relative inflammatory responses in sheep cells overexpressing of toll-like receptor 4 when stimulated with LPS[J]. PLoS One, 2012, 7(10): e47118.
[12] WANG S, SONG X, ZHANG K, et al. Overexpression of toll-like receptor 4 affects autophagy, oxidative stress, and inflammatory responses in monocytes of transgenic sheep[J]. Front Cell Dev Biol, 2020, 8: 248.
[13] LI G, LV D, YAO Y, et al. Overexpression of ASMT likely enhances the resistance of transgenic sheep to brucellosis by influencing immune-related signaling pathways and gut microbiota[J]. FASEB J, 2021, 35(9): e21783.
[14] BROWN J L, VOTH J P, PERSON K, et al. A technological and regulatory review on human-animal chimera research: the current landscape of biology, law, and public opinion[J]. Cell Transplant, 2023, 32: 9636897231183112.
[15] PU J, SCHINDLER C, JIA R, et al. BORC, a multisubunit complex that regulates lysosome positioning[J]. Dev Cell, 2015, 33(2): 176-188.
[16] GUARDIA C M, FARÍAS G G, JIA R, et al. BORC functions upstream of kinesins 1 and 3 to coordinate regional movement of lysosomes along different microtubule tracks[J]. Cell Rep, 2016, 17(8): 1950-1961.
[17] PU J, KEREN K T, BONIFACINO J S. A ragulator-BORC interaction controls lysosome positioning in response to amino acid availability[J]. J Cell Biol, 2017, 216(12): 4183-4197.
[18] STARCEVIC M, DELL A E C. Identification of snapin and three novel proteins (BLOS1, BLOS2, and BLOS3/reduced pigmentation) as subunits of biogenesis of lysosome-related organelles complex-1 (BLOC-1)[J]. J Biol Chem, 2004, 279(27): 28393-28401.
[19] 万仕成, 张梦菲, 陈文博, 等. BLOC1S1促进山羊精原干细胞增殖[J]. 生物工程学报, 2023, 39(12): 4901-4914.
WAN S C, ZHANG M F, CHEN W B, et al. BLOC1S1 promotes proliferation of goat spermatogonial stem cells[J]. Chinese Journal of Biotechnology, 2023, 39(12): 4901-4914.(in Chinese).
[20] MILLER C N, SMITH E P, CUNDIFF J A, et al. A brucella type IV effector targets the COG tethering complex to remodel host secretory traffic and promote intracellular replication[J]. Cell Host Microbe, 2017, 22(3): 317-329.
[21] JIA R, GUARDIA C M, PU J, et al. BORC coordinates encounter and fusion of lysosomes with autophagosomes[J]. Autophagy, 2017, 13(10): 1648-1663.
[22] BRIGHT M D, ITZHAK D N, WARDELL C P, et al. Cleavage of BLOC1S1 mRNA by IRE1 is sequence specific, temporally separate from XBP1 splicing, and dispensable for cell viability under acute endoplasmic reticulum stress[J]. Mol Cell Biol, 2015, 35(12): 2186-2202.
[23] PANDEY A, LIN F, CABELLO A L, et al. Activation of Host IRE1α-dependent signaling axis contributes the intracellular parasitism of brucella melitensis[J]. Front Cell Infect Microbiol, 2018, 8: 103.
[24] LIU Y, BAI X, FENG X, et al. Revolutionizing animal husbandry: breakthroughs in gene editing delivery systems[J]. Gene, 2025, 935: 149044.
[25] SHAKWEER W M E, KRIVORUCHKO A Y, DESSOUKI S M, et al. A review of transgenic animal techniques and their applications[J]. J Genet Eng Biotechnol, 2023, 21(1): 55.
[26] LEAL A F, HERRENO-PACHÓN A M, BENINCORE-FLÓREZ E, et al. Current strategies for increasing knock-in efficiency in CRISPR/Cas9-based approaches[J]. Int J Mol Sci, 2024, 25(5): 2456.
[27] XU Y N, UHM S J, KOO B C, et al. Production of transgenic Korean native cattle expressing enhanced green fluorescent protein using a FIV-based lentiviral vector injected into MII oocytes[J]. J Genet Genomics, 2013, 40(1): 37-43.
[28] HUNTER C V, TILEY L S, SANG H M. Developments in transgenic technology: applications for medicine[J]. Trends Mol Med, 2005, 11(6): 293-298.
[29] WHITELAW C B. Transgenic livestock made easy[J]. Trends Biotechnol, 2004, 22(4): 157-169.
[30] SALGADO B, RIVAS R B, PINTO D, et al. Genetically modified pigs lacking CD163 PSTII-domain-coding exon 13 are completely resistant to PRRSV infection[J]. Antiviral Res, 2024, 221: 105793.
[31] YUAN M, ZHANG J, GAO Y, et al. HMEJ-based safe-harbor genome editing enables efficient generation of cattle with increased resistance to tuberculosis[J]. J Biol Chem, 2021, 296: 100497.
[32] FENG R, ZHAO J, ZHANG Q, et al. Generation of anti-mastitis gene-edited dairy goats with enhancing lysozyme expression by inflammatory regulatory sequence using ISDra2-TnpB system[J]. Adv Sci, 2024, 5: 2404408.
[33] DENG S, LI G, ZHANG J, et al. Transgenic cloned sheep overexpressing ovine toll-like receptor 4[J]. Theriogenology, 2013, 80(1): 50-57.
[34] NKHABINDZE B Z, MAGAGULA C N, EARNSHAW D, et al. Regulatory framework for genetically modified organisms in the Kingdom of Eswatini[J]. GM Crops Food, 2024, 15(1): 212-221.
[35] SHARMA M K, LEE J, SHI H, et al. Effect of dietary inclusion of 25-hydroxyvitamin D₃ and vitamin E on performance, gut health, oxidative status, and immune response in laying hens infected with coccidiosis[J]. Poult Sci, 2024, 103(9): 104033.
[36] KOOPMANS L, SPOELDER M, BONGERS C, et al. The effect of lesser mealworm protein on exercise-induced muscle damage in active older adults: a randomized controlled trial[J]. J Nutr Health Aging, 2024, 28(5): 100204.
[37] LIU Z, JIN P, LIU Y, et al. A comprehensive approach to lifestyle intervention based on a calorie-restricted diet ameliorates liver fat in overweight/obese patients with NAFLD: a multicenter randomized controlled trial in China[J]. Nutr J, 2024, 23(1): 64.
[38] LV X, LIU Y, LIU S, et al. Metabonomics and pharmacodynamics studies of gentiana radix and wine-processed gentiana radix in damp-heat jaundice syndrome rats[J]. J Ethnopharmacol, 2024, 332: 118291.
[39] ZHU C, LIU Y, XU H, et al. Production of second-generation sheep clones via somatic cell nuclear transfer using amniotic cells as nuclear donors[J]. Theriogenology, 2025, 232: 79-86.
[40] YAO Y C, HAN H B, SONG X T, et al. Growth performance, reproductive traits and offspring survivability of genetically modified rams overexpressing toll-like receptor 4[J]. Theriogenology, 2017, 96: 103-110.
[41] 柴孟龙. 抗病转基因羊胚胎生产、移植及后代生物安全评价研究[D]. 长春: 吉林农业大学, 2012.
CHAI M L. Disease resistance transferred gene sheep embryo production, transplantation and future generations biosafety evaluation research[D]. Changchun：Jilin Agricultural University, 2012. (in Chinese)
[42] JACKSON K A, BERG J M, MURRAY J D, et al. Evaluating the fitness of human lysozyme transgenic dairy goats: growth and reproductive traits[J]. Transgenic Res, 2010, 19(6): 977-986.
[43] SMITH E P, COTTO R A, BORGHESAN E, et al. Epistatic interplay between type IV secretion effectors engages the small GTPase Rab2 in the Brucella intracellular cycle[J]. mBio, 2020, 11(2): 3350.
[44] STARR T, CHILD R, WEHRLY T D, et al. Selective subversion of autophagy complexes facilitates completion of the brucella intracellular cycle[J]. Cell Host Microbe, 2012, 11(1): 33-45.
[45] CELLI J, TSOLIS R M. Bacteria, the endoplasmic reticulum and the unfolded protein response: friends or foes?[J]. Nat Rev Microbiol, 2015, 13(2): 71-82.
[46] WU K, SEYLANI A, WU J, et al. BLOC1S1/GCN5L1/BORCS1 is a critical mediator for the initiation of autolysosomal tubulation[J]. Autophagy, 2021, 17(11): 3707-3724.
[47] WELLS K M, HE K, PANDEY A, CABELLO A, et al. Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense[J]. Elife, 2022, 11: e73625.

BLOC1S1基因过表达山羊的生产及生物安全评估

Production and Biosafety Assessment of BLOC1S1 Gene Overexpression Goats

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	严文岚, 冯宇, 薛天骐, 张子豪, 那仁毕力格, 蒋卉. 野生动物布鲁氏菌病流行现状与分析[J]. 畜牧兽医学报, 2025, 56(9): 4253-4266.
[2]	马彦男, 李政洁, 张晓, 郭贝贝, 房华, 刘子嘉, 陈时伟, 贺鹏迦, 王小龙, 陈玉林, 雷赵民. 中国奶山羊9分制体型线性评定方法学理论构建[J]. 畜牧兽医学报, 2025, 56(9): 4341-4354.
[3]	任千姿, 张佰忠, 王真勍, 王向林, 龚颖, 胡仁科, 浦亚斌, 苏鹏, 李业芳, 马月辉, 李昊帮, 蒋琳. 基于全基因组重测序对武雪山羊的遗传进化分析[J]. 畜牧兽医学报, 2025, 56(8): 3787-3801.
[4]	冯锦涛, 闫炎, 宗俊霖, 胡宇晴, 李晓蕊, 关伟军, 李耀坤. 山羊脐带间充质干细胞分离培养与治疗小鼠药物性肝损伤[J]. 畜牧兽医学报, 2025, 56(8): 4053-4064.
[5]	刘爱军, 黄晓兵, 张传亮, 张红丽. 布鲁氏菌与宿主天然免疫信号通路相互作用的研究进展[J]. 畜牧兽医学报, 2025, 56(4): 1561-1574.
[6]	尤留超, 尹号, 陶政宇, 黄蓉, 付磊, 储岳峰. 布鲁氏菌毒力因子与胞内存活机制研究进展[J]. 畜牧兽医学报, 2025, 56(4): 1575-1593.
[7]	王浩宇, 马克岩, 李讨讨, 栗登攀, 赵箐, 马友记. 基于简化基因组测序评估小骨山羊群体遗传多样性和群体结构[J]. 畜牧兽医学报, 2025, 56(3): 1170-1179.
[8]	布威海丽且姆·阿巴拜科日, 艾合麦提尼亚孜·艾合麦提江, 乃比江·麦图荪, 单文娟. 塔里木马鹿抗氧化基因PRDX1的功能初探[J]. 畜牧兽医学报, 2025, 56(3): 1216-1230.
[9]	杨晓雯, 宁文晴, 周师众, 袁雅琴, 侯雪新, 丁家波. 一种羊种布鲁氏菌复方新诺明耐药株荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2025, 56(3): 1465-1472.
[10]	习海娇, 李金泉, 张燕军, 王瑞军, 吕琦, 梅步俊, 王娜, 苏蕊, 王志英. 显性效应对内蒙古绒山羊产绒量和绒纤维直径育种值估计准确性的影响[J]. 畜牧兽医学报, 2025, 56(2): 571-581.
[11]	冯婧, 盛辉, 张效生, 郭晓飞, 姚大为, 李玉鹏, 陈龙宾, 张金龙. miR-665靶向BCL2L11调控武安山羊成肌细胞增殖[J]. 畜牧兽医学报, 2025, 56(2): 582-590.
[12]	李枝卉, 罗金林, 谢虹, 李明坤, 林杨, 张宇姣, 徐引弟, 陈焕春, 蔡旭旺, 徐晓娟. 副猪格拉瑟菌OppA过表达株的构建及其对小鼠免疫保护性研究[J]. 畜牧兽医学报, 2025, 56(2): 860-869.
[13]	王晓飞, 王勃森, 卫梦瑶, 姜璐瑶, 徐刚刚, 刘佳欣, 马应天, 王丽, 宋宇轩, 张磊. 羊奶改善糖尿病模型小鼠肝、肾病理变化的作用研究[J]. 畜牧兽医学报, 2025, 56(2): 870-882.
[14]	于凤姣, 刘开东, 宋伟杰, 柳楠, 李和刚, 赵金山, 高霄霄, 贺建宁. miR-137靶向MITF调控山羊黑色素细胞生成黑色素的机制研究[J]. 畜牧兽医学报, 2025, 56(1): 189-200.
[15]	吴少强, 刘雨帆, 韦义荣, 黄艳娜, 蒋钦杨. 白藜芦醇通过PROX1/SIRT1信号通路调控山羊肌纤维类型转化的机制研究[J]. 畜牧兽医学报, 2025, 56(1): 201-212.