Thiết kế probe để khai thác gen mã hóa pectinesterase từ dữ liệu giải trình tự DNA metagenome và đồng biểu hiện gen gpecs1 của vi khuẩn trong dạ cỏ của dê với chaperone pG-KJE8 trong Escherichia coli

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Tác giả: Thị Huyền Đỗ, Tùng Lâm Lê, Khánh Hoàng Việt Nguyễn, Thủy Tiên Nguyễn, Thị Lan Phùng, Thu Nguyệt Phùng, Nam Hải Trương

Ngôn ngữ: vie

Ký hiệu phân loại: 615.35 Enzymes

Thông tin xuất bản: Sinh học, 2018

Mô tả vật lý: 84-91

Bộ sưu tập: Metadata

ID: 492065

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This article introduces the steps of constructing and using probe to exploit the gene encoding pectinesterase from metagenome DNA sequencing data by next generation gene sequencing tools. Probe was, used to exploit and select the gene encoding for pectinesterase from the metagenome DNA sequences of bacteria in goat rumen and thereby select a sequence to express in E. coli. According to the CAZy, classification system, pectinesterase belongs to the family of carbohydrates esterases CE8 is an enzyme that has many applications in the food processing industry, environmental treatment, animal feed processing and medicine. As the results, 3 sequences of CE8 was retrieved from CAZy database and one probe was designed, this probe length was 367 amino acids contained all the conserved amino acid residues: 200 conserved residues in all sequence, 72 residues similar in almost sequences and residues conserved in many sequences and homologus
choosed highest alkalinity index. Using the probe designed, we filtered four coding sequences for pectinesterase from metagenome DNA sequencing data of bacteria in goat rumen. Spatial structure estimation with Phyre2 has only one sequencing (code 46301) with 100% sequence identity and 90% query coverage with pectinesterase. A artificial gene were synthesized and inserted into the vector pET22b (+) at the NcoI, XhoI to co-express with chaperone pG-KJE8 in E. coli. The recombinant pectinesterase enzyme is expressed in soluble form and has a pectin substrate biodegradation activity. The results demonstrate that using probe for gene extraction is feasible.Trình bày các bước xây dựng và sử dụng probe để khai thác gen mã hóa pectinesterase từ dữ liệu giải trình tự DNA metagenome bằng công cụ giải trình tự gen thế hệ mới. Dựa trên 3 trình tự thuộc họ CE8 tìm kiếm được từ cơ sở dữ liệu đã xây dựng được một probe có độ dài 367 amino acid, trong đó có 200 amino acid hoàn toàn giống nhau, 72 vị trí giống nhau ở đa số các trình tự và 47 vị trí giống nhau ở một số trình tự. Kết quả ước đoán cấu trúc không gian bằng Phyre2 đã chọn được duy nhất một trình tự (mã số 46301) có độ tin cậy, độ bao phủ lần lượt là 100% và 90% với pectinesterase. Gen đã được đặt tổng hợp và đưa vào vector pET22b(+) tại vị trí NcoI, XhoI để đồng biểu hiện với chaperone từ vector pG-KJE8 trong E. coli. Pectinesterase được biểu hiện ở dạng tan và có hoạt tính sinh học thủy phân cơ chất pectin.

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