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 出版日期:2025-09-23 发布日期:2025-09-30
通讯作者: 朱海鲸,华进联 E-mail:wcl1996@nwafu.edu.cn;shicheng_wan@nwafu.edu.cn;haijingzhu@yulinu.edu.cn;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
第一联系人：
王聪亮和万仕成为同等贡献作者
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 Online:2025-09-23 Published:2025-09-30
Contact: ZHU Haijing, HUA Jinlian E-mail:wcl1996@nwafu.edu.cn;shicheng_wan@nwafu.edu.cn;haijingzhu@yulinu.edu.cn;jinlianhua@nwsuaf.edu.cn

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.

图/表 6

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	退火温度/℃ Tm	产物长度/bp Products length
BLOC1S1-F	ttctagagctagcGAATTCGATGCTGTCCCGCTTGCTGAA	65	418
BLOC1S1-R	ccttcgcggccgcGGATCCGCTAGGAGGGGGCCGACTGCA
CMV/BLOC1S1-F	AGTGAACCGTCAGATCGCCT	58	442
CMV/BLOC1S1-R	ACTGCAGCTGCCCTTTGTAG
Q-BLOC1S1-F	AGGAAGCTTGACCATGAGGTG	60	110
Q-BLOC1S1-R	TCCTTGAGTGCCTGGTTGAAG
Q-β-ACTIN-F	TGATATTGCTGCGCTCGT	60	196
Q-β-ACTIN-R	CTTGAGGGTCAGGATGCC

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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

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