七鳃鳗Lja-SHP2分子鉴定、重组表达及免疫学研究

doi:10.16288/j.yczz.19-260

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Hereditas(Beijing) ›› 2020, Vol. 42 ›› Issue (2): 183-193.doi: 10.16288/j.yczz.19-260

• Research Article • Previous Articles Next Articles

Identification, recombinant expression and immunological study of Lja-SHP2 in Lampetra japonica

Xin Li¹(), Chengming Qu¹, Yinglun Han^1,², Xin Liu^1,²(), Qingwei Li^1,²()

^1. Research Center of Lamprey, School of Life Sciences, Liaoning Normal University, Dalian 116029, China
^2. Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University, Dalian 116034, China

Received:2019-09-02 Revised:2019-11-15 Online:2020-01-09 Published:2020-01-08
Supported by:
the National Natural Science Foundation of China(31801973);the Liaoning Provincial Department of Education Research Fund Key Project(LZ201783601)

Abstract

Abstract:

The protein tyrosine phosphatase SHP2 of higher vertebrates, encoded by ptpn11 gene, catalyzes the dephosphorylation of tyrosine phosphorylation site, and plays regulatory roles in various signaling pathways by cooperating with other protein tyrosine kinase. Previous studies have shown that SHP2 plays an important role in the activation and signal transduction of T and B cells in higher vertebrates. To study the role of a SHP2 homologous molecule of lampreys (Lja-SHP2) in immune response, we cloned and expressed the open reading frame sequence of Lja-SHP2 gene in prokaryotic expression vector pET-32a. The recombinant protein was successfully expressed in E. coli and the rabbit-derived polyclonal antibody was prepared. Lampetra japonica were immunized with mixed bacteria, and the mRNA and protein of Lja-SHP2 in immune-related cells and tissues were detected by real-time quantitative PCR and Western blotting after immunization. The Lja-SHP2 mRNA and protein were not significantly affected in leukocytes and supraneural myeloid bodies, but up-regulated significantly in gill tissues (P<0.05) after challenged by mixed bacteria, which indicated that Lja-SHP2 mainly participates in the immune response of gill tissues after mixed bacteria stimulation. To further investigate whether Lja-SHP2 level was affected in three lymphocyte subsets, the B-cell mitogen lipopolysaccharide (LPS) and T-cell mitogen phytohaemagglutinin (PHA) were employed to boost the immune response in L. japonica. LPS immune stimulation increased Lja-SHP2 in leucocytes significantly compared with the control group, and but had a marginal effect on Lja-SHP2 expression in gills and supraneural myeloid bodies. PHA immune stimulation could up-regulate Lja-SHP2 level in leukocytes, gill tissues and supraneural myeloid bodies. The change of Lja-SHP2 was especially dramatical in leukocytes, which was about 2.5 times higher than that in the control group, suggesting that Lja-SHP2 is involved in the lamprey immune response mediated by PHA. Consistent with the previous finding that PHA could induce the activation of VLRA⁺ lymphocytes, our results showed that Lja-SHP2 might be included in the immune response of VLRA⁺ lymphocytes mediated by PHA in gills. This research will benefit exploring the functions of Lja-SHP2 in the immune response of lamprey and will provide clues for understanding the phylogenesis of SHP2 molecular family, and its roles in the early occurrence and evolution of adaptive immune system in higher vertebrates.

Key words: Lampetra japonica, Lja-SHP2, antibody preparation, immune response

Xin Li, Chengming Qu, Yinglun Han, Xin Liu, Qingwei Li. Identification, recombinant expression and immunological study of Lja-SHP2 in Lampetra japonica[J]. Hereditas(Beijing), 2020, 42(2): 183-193.

Figures/Tables 6

Table 1

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Fig. 4

Supplemental Fig. 1

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引物名称	引物序列(5′→ 3′)	用途
ORF-outer	F：AGTCCCATCGCAGGCTC	ORF区扩增
ORF-outer	R: TAAGTCTGCCGCTACCCG	ORF区扩增
ORF-inner	F: ATGACCTCCCAAAGATGGTT	ORF区扩增
ORF-inner	R: TCAACAGTCTGAGGGCCTT	ORF区扩增
Lja-SHP2-EXP	F: CCGGAATTCATGACCTCCCAAAGATGGTT	表达载体构建
Lja-SHP2-EXP	R: CCCAAGCTTTCAACAGTCTGAGGGCCTT	表达载体构建
Lja-SHP2-qRT-PCR	F: GTTCCACCCGCACATCAC	qRT-PCR扩增目的基因
Lja-SHP2-qRT-PCR	R: CGCACGGACAAGGTGAAGT	qRT-PCR扩增目的基因
gapdh-qRT-PCR	F: GGCATCCAAGGGTGAACT	qRT-PCR扩增内参基因
gapdh-qRT-PCR	R: CGGGTGCTGTAGCCAAAC	qRT-PCR扩增内参基因

Identification, recombinant expression and immunological study of Lja-SHP2 in Lampetra japonica

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