弓形虫TR与ROP5双基因缺失株的构建及其生物学功能研究

doi:10.11843/j.issn.0366-6964.2025.07.033

摘要/Abstract

摘要：

为了探究弓形虫TR与ROP5在抗宿主ROS损伤中是否具有协同作用，本研究利用CRISPR/Cas9技术成功构建了TR与ROP5双基因缺失株(TR-ROP5-KO)弓形虫。通过体外增殖、入侵及小鼠体内毒力试验发现，与RH株、TR基因缺失株(TR-KO)及ROP5基因缺失株(ROP5-KO)相比，TR-ROP5-KO株在体外细胞中入侵、增殖能力及小鼠体内毒力作用减弱，表明弓形虫TR与ROP5基因双缺失不仅导致虫体在体外细胞中的生存活力减弱，还降低对宿主的毒力作用；通过检测TR/ROP5基因缺失株的氧化应激水平以及感染RAW264.7细胞的活性氧(ROS)水平发现，TR-ROP5-KO株引发的氧化应激水平虽然显著高于RH株(P < 0.05)，但与TR-KO株无显著差异(P > 0.05)；利用RT-qPCR检测TR/ROP5基因缺失株感染RAW264.7细胞的NF-κB、IL-12、IFN-γ mRNA水平和ELISA检测小鼠血清中IL-12的水平发现，TR-ROP5-KO株感染组虽显著高于RH株感染组(P < 0.05)，但却未显著高于TR-KO株感染组(P > 0.05)，表明弓形虫的TR与ROP5在抗宿主ROS损伤中不存在协同作用。本研究构建TR与ROP5的双基因缺失株的方法探究两者在抗宿主ROS损伤中并无协同作用，为后续解析弓形虫多个基因功能的研究提供了方法依据，为弓形虫免疫逃避机制的研究提供了基础材料。

关键词: 刚地弓形虫, CRISPR/Cas9, TR, ROP5, ROS

Abstract:

To explore whether the TR and ROP5 of Toxoplasma gondii have a synergistic effect on anti-host ROS damage, we used CRISPR/Cas9 technology in this study to successfully obtain the TR and ROP5 double gene knockout strain (TR-ROP5-KO). By biological activity analysis, we discovered that the invasion, proliferation ability in vitro, and virulence in vivo of TR-ROP5-KO strain were lower than those of RH strain, TR gene deletion strain (TR-KO) and ROP5 gene deletion strain (ROP5-KO). These results indicated that the simultaneous deletion of TR and ROP5 in Toxoplasma gondii not only lead to a decrease in the viability of the parasites in vitro cells, but also reduce the virulence of toxoplasma in the host. The results of the oxidative stress levels of T. gondii and their effects on ROS levels in mouse macrophages cells showed that the level of oxidative stress induced by TR-ROP5-KO strain was significantly higher than by RH strain (P < 0.05), but there was no differences relative to TR single gene deletion strain (P > 0.05). The mRNA levels of NF-κB, IL-12 and IFN-γ in mouse macrophages infected with TR/ROP5 single and double gene deletion strains were detected by RT-qPCR, and the levels of IL-12 in serum of mice was also detected by ELISA. The results suggested that the TR-ROP5-KO infection group was significantly higher than RH group (P < 0.05), but the TR-ROP5-KO infection group was not significantly higher than the TR-KO infection group (P > 0.05). The results indicated that TR and ROP5 genes have no synergistic effect on resisting the ROS damage of host. In this study, the method of constructing double-gene deletion strains of TR and ROP5 showed that the two genes had no synergistic effect on the anti-ROS damage of the host, which provided the method basis for the subsequent research on the function of multiple genes of Toxoplasma gondii and the basic material for the research on the immune escape mechanism of Toxoplasma gondii.

Key words: Toxoplasma gondii, CRISPR/Cas9, TR, ROP5, ROS

中图分类号:

S852.729

耿小玲, 李瑞芳, 徐卫兵, 杜晶莹, 张曼玉, 孙卿, 蒋蔚, 米荣升, 陈兆国, 王权. 弓形虫TR与ROP5双基因缺失株的构建及其生物学功能研究[J]. 畜牧兽医学报, 2025, 56(7): 3408-3422.

GENG Xiaoling, LI Ruifang, XU Weibing, DU Jingying, ZHANG Manyu, SUN Qing, JIANG Wei, MI Rongsheng, CHEN Zhaoguo, WANG Quan. Construction and Biological Function Research of TR and ROP5 Double Gene Deletion Strains of Toxoplasma gondii[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3408-3422.

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序号 No.	名称 Name	引物的序列(5′→3′) Sequence	质粒名称 Plasmid	目的 Purpose
1	gRNA1-F	gcttctcatgcccgctgcgtGTTTTAGAGCTAGAAATAGC	pSAG1::CAS9-U6::sgROP5-1	PCR扩增片段1
	gRNA2-F	tgggggatgtatccgtgttgGTTTTAGAGCTAGAAATAGC	pSAG1::CAS9-U6::sgROP5-2
	CAS-1R	AAAAGGCAACGGATGTAAAAG	pSAG1::CAS9-U6::sgROP5
2	CAS-2F	CTTTTACATCCGTTGCCTTTTCC	pSAG1::CAS9-U6::sgROP5	PCR扩增片段2
2	CAS-2R	GCCGTTTGTCTCGATCAGAGG	pSAG1::CAS9-U6::sgROP5
3	CAS-3F	CCTCTGATCGAGACAAACGGC	pSAG1::CAS9-U6::sgROP5	PCR扩增片段3
	3gRNA1-R	acgcagcgggcatgagaagcAACTTGACATCCCCATTTACCAG	pSAG1::CAS9-U6::sgROP5-1
	3gRNA2-R	caacacggatacatcccccaAACTTGACATCCCCATTTACCAG	pSAG1::CAS9-U6::sgROP5-2
4	DgRNA-F	GCTTCCTCCCTGTGCGCT		鉴定gRNA1是否插入质粒
4	DgRNA-R	GGAACAAAAGCTGGAGCTCAAAAAAG
5	U6-F	$\underline{{\rm{CGAATTG}}}$GGTACCCAAGTAAGCAGAAGCACGCTG(Kpn Ⅰ)	pSAG1::CAS9-U6::sgROP5	扩增ROP5含Kpn Ⅰ和Xho Ⅰ酶切位点的gRNA2片段
5	U6-R	$\underline{{\rm{TCGAC}}}$CTCGAGAATTAACCCTCACTAAAGG(Xho Ⅰ)		扩增ROP5含Kpn Ⅰ和Xho Ⅰ酶切位点的gRNA2片段
6	DgRNA-2F	AAAACGACGGCCAGTGAG		鉴定gRNA2是否插入质粒
	DgRNA-2R	GAAAAGGCAACGGATGTAA

序号 No.	名称 Name	引物的序列(5′→3′) Sequence	目的 Purpose
1	ROP5 5′KO-F	ACATCGAGGTGGACGTGGAAATATA	扩增ROP5 5′UTR
1	ROP5 5′KO-R	CTGGGAGATGTTGTGAGGTCTTGGT
2	ROP5 3′KO-F	GAAAACGGCTCTGCCAACGGGGCGT	扩增ROP5 3′UTR
2	ROP5 3′KO-R	GCTGCCTCTGTGGATCTCTCTAAAC
3	CAT-F	accaagacctcacaacatctcccagACATGTCGGGCCCCCCCT	扩增CAT抗性片段
3	CAT-R	acgccccgttggcagagccgttttcTCTAGAACTAGTGGATCCCCC
4	pROP5:CAT-F	gtttagagagatccacagaggcagcGTCGTACAGTTGACGTCGA	扩增质粒骨架
4	pROP5:CAT-R	tatatttccacgtccacctcgatgtGACAACTTTTCTATACAAAGTTGATAG
5	ROP5-PCR-1-F	ACATCGAGGTGGACGTGGAAATATA	判断5′UTR与CAT连接处是否连上
5	ROP5-PCR-1-R	acgccccgttggcagagccgttttcTCTAGAACTAGTGGATCCCCC	判断5′UTR与CAT连接处是否连上
6	ROP5-PCR-2-F	ACATCGAGGTGGACGTGGAAATATA	判断3′UTR与CAT连接处是否连上
	ROP5-PCR-2-R	acgccccgttggcagagccgttttcTCTAGAACTAGTGGATCCCCC

序号 No.	名称 Name	引物的序列(5′→3′) Sequence	作用 Role	目的 Purpose
1	TR-F	CGAGTTCTGTATCAGAGGCTGTCCG	扩增TR基因的820 bp	判断TR基因是否缺失
1	TR-R	TGTCGAGTCAGCTCAAACGAGAGC
2	ROP5-F	CGGTGACCGATCTGTCGTATTTTTA	扩增ROP5基因的901 bp	判断ROP5基因是否缺失
2	ROP5-R	AGGTTGTCCTGTTGATAGGCTGCTC
3	CAT-F	ACATGTCGGGCCCCCCCT	扩增CAT抗性片段的1 546 bp	判断虫体是否具有CAT抗性
3	CAT-R	TCTAGAACTAGTGGATCCCCC
4	DHFR-F	AAGCTTCGCCAGGCTGTA	扩增DHFR抗性片段的3 163 bp	判断虫体是否具有DHFR抗性
	DHFR-R	GGAATTCATCCTGCAAGTGC

引物名称 Name	核苷酸序列(5′→3′) Nucleotide sequence	PCR产物长度/bp PCR product length
β-Actin-F	AGAGGGAAATCGTGCGTGAC	138
β-Actin-R	CAATAGTGATGACCTGGCCGT
IL-12-F	GGAAGCACGGCAGCAGAATA	180
IL-12-R	AACTTGAGGGAGAAGTAGGAATGG
IFN-γ-F	TCAAGTGGCATAGATGTGGAAGAA	92
IFN-γ-R	TGGCTCTGCAGGATTTTCATG
NF-κB-F	TGGCTACTATGAGGCTGACC	787
NF-κB-R	GTTGATGGTGCTGAGGGAT

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