Effects of ORFV118 on Cell Cycle,Apoptosis and the Immune-related Cytokines in Goat Testicular Sertoli Cells

doi:10.7668/hbnxb.20192480

Abstract

Abstract:

This aim of study was to explore the role of ORFV118 in regulating cell cycle,apoptosis and the immune-related cytokines(IL-1β,IL-6,IFN-γ and TNF-α)in goat testicular sertoli cells.We cloned the whole sequence of ORFV118 and constructed an expression vector of pEGFP-ORFV118.The expression of ORFV118 protein was then identified by using Western Blot method after the transfection of goat testicular sertoli cells with pEGFP-ORFV118.Following which,a flow cytometer equipment was used to measure the effect of ORFV118 expression on cell cycle and apoptosis of goat testicular sertoli cells,and also on regulating the expression of genes related to cell cycle,including CDK2 and P21,and genes relative to apottosis,including Bax,Bcl-2,Caspase3,Caspase7,P53 and BCL2L11.The expression of immune-related cytokines,including IL-1β,IL-6,IFN-γ and TNF-α,was detected by ELISA method and RT-qPCR after ORFV118 expression in goat testicular sertoli cells.The results showed that a total length of 309 bp ORFV118 gene sequence was cloned successfully.The cell's DNA synthesis phase(S phase)was inhibited and the late stage DNA synthesis(G2/M phase)was promoted by the expression of ORFV118,and the expression of CDK2 mRNA was decreased.The cell apoptosis blocked after ORFV118 expression,and the expression of Caspase3,Caspase7 and SOCS2 mRNA decreased.The immune-related cytokines,including IL-1β and IFN-γ,was inhibited in goat testis cells,while the expression of IL-6 and TNF-α were promoted.

Key words: Goat, Orf virus, ORFV118 protein, Cell cycle, Cell apoptosis, Cytokines

WANG Xianjun, XIANG Hua, ZHANG Xumei, REN Yupeng, ZHU Jiangjiang. Effects of ORFV118 on Cell Cycle,Apoptosis and the Immune-related Cytokines in Goat Testicular Sertoli Cells[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 222-231. doi: 10.7668/hbnxb.20192480.

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Figures/Tables 10

Tab.1 Primer sequences of cytokine

引物名称 Primer	引物序列(5'—3') Primer sequence(5'—3')	登录号 Accession No.	退火温度/℃ TM	扩增产物大小/bp PCR product length
IL-1β	S:TCACCAGCTCTACAACAAA A:AGGTCATCATCACGGAAG	KY038171.1	60	105
IL-6	S:GCTTCCAATCTGGGTTCA A:AGTTTCCTGATTTCCCTC	AB028635.1	60	114
IFN-γ	S:CAGAGCCAAATTGTCTCCTTC A:ATCCACCGGAATTTGAATCAG	AF020684.1	60	167
TNF-α	S:CCAGAGGGAAGAGCAGTCC A:GGCTACAACGTGGGCTACC	MH180383.1	60	111
GAPDH	S: GCAAGTTCCACGGCACAG A: TCAGCACCAGCATCACCC	XM_005680968.3	62	118

Tab.2 Primers sequences of cell cycle and apoptosis-related genes

目的基因 Gene	引物序列(5'—3') Primer sequence(5'—3')	登录号 Accession No.	退火温度/℃ TM	产物长度/bp Products length
BaX	S:CGCATTGGAGATGAATTGGACAGTAAC	KJ782303.1	60	128
	A:CAGTTGAAGTTGCCGTCGGAAAAC
Bcl-2	S:TGTGGATGACCGAGTACCTGAACC	KJ782304.1	60	127
	A:AGAGACAGCCAGGAGAAATCAAACAG
Caspase3	S:ACGCATATTCTACAGCACCTGGTTAC	JN986829.1	60	82
	A:ATCTCACAAAGAGCCTGGATGAACC
Caspase7	S:GGAACAGATGGCAAGACAGCAATAAAG	XM_018041622.1	60	101
	A:GCCTGAATGAAGAAGAGTTTGGGTTTC
BCL2L11	S:AACCTTCCGATGTAAGTTCTGAGTGTG	NM_001075310.1	60	117
	A:TGTCTTGCCGCTCTGTCTGTAAAG
P53	S:AGTGTGGTGGTGCCCTATGAGTC	X81704.1	60	137
	A:GAGTCTTCCAGTGTGATGATGGTGAG
SOCS2	S:TTCGCATTCAGATTACCTACT	XM_018047627.1	60	192
	A:CCTGTCCGCTTATCCTTG
PARP1	S:TCGCATATCAGCAAGTTACCCAAGG	XM_027564618.1	60	102
	A:CACCTCCACACCATCCACAGTAATAC
CDK2	S:GCACTCACTGGCATTCCTCTTCC	NM_001285608.1	60	140
	A:ATGGACCCATCTGCGTTGATAAGC
P21	S:GGTAGAGTTGGCTTTATGGGACACAG	NM_176645.3	60	114
	A:TCAGGGCTATCAATGGAGAAACACATC
UXT	S:GCAAGTGGATTTGGGCTGTAAC	JN711468.1	60	180
	A:ATGGAGTCCTTGGTGAGGTTGT

Fig.1 Amplification,cloning and identification of ORFV118 gene
A.ORFV118 gene amplified by PCR;B.Recombinant plasmid pMD19T-ORFV118 amplified by PCR.M.2 000 bp DNA ladder;P.Positive product;N.Negative product;1.ORFV118 gene.The same as Fig.4.

Fig.2 Bioinformatics analysis of ORFV118 protein
A.Hydrophobicity analysis of ORFV118 protein;B.Signal peptide prediction of ORFV118 protein;C.Transmembrane domain analysis of ORFV118 protein;D.Conserved domain prediction of ORFV118 protein;E.Secondary structure prediction of ORFV118 protein;F.Tertiary structure prediction of ORFV118 protein;G.Glycosylation site prediction of ORFV118 protein;H.Phosphorylation site prediction of ORFV118 protein;I.Predicted subcellular localization of ORFV118 protein.

Fig.3 Enzyme identification results of pEGFP-ORFV118
M.5000 DNA Ladder;1.pEGFP-ORFV118 digested by Hind Ⅲ and BamHⅠ.

Fig.4 mRNA analysis of pEGFP-ORFV118

Fig.5 Western Blot identification of ORFV118 protein

Fig.6 Effect of ORFV118 on IL-1β,IL-6,IFN-γ and TNF-α expression
Different lowercase letters indicate significant difference at the level of 0.05 between the treatment and the control; Different capital letters indicate extremely significant difference at the level of 0.01 between the treatment and the control.The same as Fig.7—8.

Fig.7 Effect of ORF118 protein transfection on cell cycle of goat testicular sertoli cells
A.Effect of pEGFP-N1 on cell cycle of goat primary testis;B.Effect of pEGFP-ORFV118 on cell cycle of goat primary testis; C.Cell cycle results of different groups;D.Effect of pEGFP-ORFV118 on the expression of cell cycle-related genes.

Fig.8 Effect of ORF118 protein transfection on cell apoptosis of goat testicular sertoli cells
A.Effect of pEGFP-N1 on apoptosis of goat testicular sertoli cells;B.Effect of pEGFP-ORFV118 on apoptosis of goat testicular sertoli cells C.Effect of RT-qPCR detect apoptosis-related genes.

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