新疆南疆和田羊、卡拉库尔羊毛囊KAP7基因的原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2017.12.003

Abstract

Abstract:

This study was aimed to prepare polyclonal antibodies of wool follicle keratin associated protein 7 (KAP7) gene of three local variety (strain) sheep of Plain-type Hetain sheep, Mountain-type Hetain sheep and Karakul sheep in Southern Xinjiang, to provide the basis for exploring influences of follicle KAP7 gene to coarse wool quantity and quality. This study took the skin wool follicle samples from three local variety (strain) sheep in Southern Xinjiang, extracted total RNA from the skin wool follicle samples, obtained cDNA through reverse transcription, then amplified the wool follicle KAP7 gene fragments by PCR. We constructed the pMD19-T-KAP7 recombination cloning plasmids, and tested the correction of the plasmids by PCR assay, enzyme digest and sequence analysis, then constructed the prokaryotic expression recombination plasmids of pET-28a(+)-KAP7,expressed them in E.coli BL21 (DE3) cells. SDS-PAGE electrophoresis was employed to detect the prokaryotic expression wool follicle KAP7 protein. We obtained the target proteins by extraction, purification and reclaim. The polyclonal antibodies serum were got from the rabbits which were immunifaciented with the purification KAP7 protien. The antibodies titer were detected by indirect ELISA. The results showed that there were 99% homology among the Merino sheep with the mountain-type and plain-type Hetian sheep, but there was a mutation at the 22nd base pair of KAP7 gene of plain-type Hetian sheep, in which it conversed from G to C, and this made the 8th amino acid of KAP7 protein to converse from Gly to Arg. For the mountain-type Hetian sheep, there was a mutation from A to C at the 70th base pair, this changed the 24th amino acid of KAP7 protein from Thr to Ala. The homology of wool follicle KAP7 gene was 100% between Karakul sheep and Merino sheep, so there was no differences on the amino acids sequences between them. The prokaryotic expression plasmids pET-28a(+)-KAP7 of wool follicle KAP7 gene could be expressed in E.coli BL21 cells, and the wool follicle KAP7 proteins were about 10 ku. The positive polyclonal antibodies serum of the sheep wool follicle KAP7 proteins, which were obtained from immunized rabbits, had immunological activity and specificity, and the antibodies titer could be up to 1:10 000.

Key words: Hetian sheep; Karakul sheep; keratin associated protein; wool KAP7 gene; wool follicle

CLC Number:

LI Zhi-gang, WANG Li, LI Shu-wei. Prokaryotic Expression and Preparation Polyclonal Antibody of Wool Follicle KAP7 Gene of Hetian Sheep and Karakul Sheep in Southern Xinjiang[J]. , 2017, 44(12): 3410-3417.

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Prokaryotic Expression and Preparation Polyclonal Antibody of Wool Follicle KAP7 Gene of Hetian Sheep and Karakul Sheep in Southern Xinjiang

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