Study on Characteristics of Chemokine CXCL10 Gene Cloned from cDNA Expression Library of Ujumqin Sheep

P F Hu,Q J Zhao,X C Li,Y H Ma,N Lei,X Y Lan,W J Guan

doi:10.1155/2013/217942

Abstract

Chemokines were a major regulator of body's inflammatory and immune responses. In this study, the cDNA fragment of chemokine CXC ligand 10 (CXCL10) was cloned from the Ujumqin sheep ear marginal tissue cDNA expression library; the CXCL10 gene had 103 amino acids and a molecular weight of 11.47 kDa, and it shared a high homology among cattle, sheep, and goat, while a low homology compared with mouse. The CXCL10 protein had 4 conservative cysteine residues, located in 28, 30, 55, and 72 sites. The expression pattern and intracellular distribution of recombinant CXCL10 proteins in Ujumqin sheep fibroblast cells showed that there were green fluorescence signals both in cytoplasm and nucleolus after 24 h of transfection, the number of positive cells was increased with time, the peak level of fluorescence signal was reached after 48 h of transfection and the transfection efficiency was 33.3%; there was a significant decrease in fluorescence intensity after 72 h of transfection. Expression of recombinant CXCL10 gene in Escherichia coli had a time- and temperature-dependency on the amount of protein expression, and a small quantity of inducer was needed.

Highlights

Ujumqin sheep, a larger version of the Mongolian, was found in Inner Mongolia, China
Titers of the unamplified and amplified library were determined by counting the number of colonies according to the protocol of cDNA Library Construction Kit; the results showed that unamplified and amplified library had a titer of 8.24 × 106 pfu/mL and 1.60 × 1010 pfu/mL, respectively
On the basis of the high identity, it was concluded that the cDNA isolated was the cDNA encoding the Ujumqin sheep CXC ligand 10 (CXCL10) gene

Summary

Introduction

A larger version of the Mongolian, was found in Inner Mongolia, China. These sheep have a type of coarse wool commonly called “carpet wool” and have an ability to deposit fat in the tail; they could adapt to the unfavourable local environmental conditions of the north and northwest pastoral grasslands. Our group have been engaged in the study of molecular mechanism on the growth and development of sheep for many years; a series of genes were found, such as NADH dehydrogenase subunit 2(ND2), subunit 4 (ND4) [6], TSK21 [7], cytochrome c oxidase subunit VIa polypeptide 1-like gene GU585577); we are paying more attention to chemokines of Ujumqin sheep because of their multiple biological functions especially in the mechanism of development, prevention and treatment of disease

Methods

Results

Conclusion