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Trypanosoma brucei phenylalanyl-tRNA synthetase preparation method

A technology of trypanosoma brucei and phenylalanyl is applied in the field of preparation of trypanosoma brucei phenylalanyl tRNA synthetase, and can solve the problem of low expression efficiency and expression purification of trypanosoma brucei phenylalanyl tRNA synthetase The steps are cumbersome and other problems, and the steps are simple and the purification effect is good.

Active Publication Date: 2010-01-27
泰州新生源生物医药有限公司
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Problems solved by technology

[0004] Find through literature search to prior art, Nina Moor has published " Cloning and Expression of Human Phenylalanyl-tRNA Synthetase in Escherichia " in " Protein Expression and Purification " (" Protein Expression and Purification ") 2002 24th pages 260~267 pages coli: Comparative Study of Purified Recombinant Enzymes" ("Clone and Expression of Human Phenylalanyl tRNA Synthetase in Escherichia coli: Study and Comparison of Purified Recombinant Enzymes"), the article gives the expression and purification of human phenylalanyl tRNA synthesis Enzyme method, but the expression and purification steps of this method are cumbersome, and the expression efficiency of Trypanosoma brucei phenylalanyl tRNA synthetase is low

Method used

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Embodiment

[0034] Step 1, obtaining the α subunit and β subunit genes of Trypanosoma brucei PheRS

[0035] 1. Acquisition of the α subunit gene of PheRS

[0036] (1) Take 20 μg of whole genome DNA of Trypanosoma brucei as a template, amplify the target gene fragment by PCR method, and the upstream and downstream primers used for amplification are primers purified by PAGE, wherein,

[0037] Upstream (Primer F): 5'ATGAGTACCATGGAGAACACCATTCT,

[0038] Downstream (Primer R): 5'AAAACGCATTAGCTTTGAGTTCC;

[0039] (See SEQ ID NO: 1 for the upstream sequence, and SEQ ID NO: 2 for the downstream sequence).

[0040] (2) PCR reaction was carried out with Pfu polymerase, in which the template was genomic DNA, the primers used were Primer F and Primer R, pre-denatured at 98°C for 2 min, denatured at 95°C for 1 min, annealed at 55°C for 1 min, and extended at 68°C for 3 min. 35 cycles, and finally extended at 72°C for 10 min; add 5× loading buffer to the PCR-amplified product, and use 1% agarose gel...

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Abstract

The invention relates to a trypanosoma brucei phenylalanyl-tRNA synthetase preparation method, belonging to the technical field of biology and the preparation method comprises the following specific steps: separately designing primers, amplifying the alpha subunit and beta subunit genes of trypanosoma brucei phenylalanyl-tRNA synthetase; cloning the alpha subunit and beta subunit genes into prokaryotic expression vector pET21a(+), building pET21a(+)-PheRS alpha and pET21a(+)-PheRS beta expression vector; subcloning the operon of alpha subunit into pET21a(+)-PheRS beta, forming recombinant co-expression vector pET21a(+)-PheRS alpha beta; transforming the recombinant co-expression vector pET21a(+)-PheRS alpha beta into colibacillus BL21(DE3)RIPL, inducting expression with IPTG; collecting the supernate of the lysed bacteria solution, purifying proteins and obtaining the trypanosoma brucei phenylalanyl-tRNA synthetase. The invention builds the recombinant plasmid of the trypanosoma brucei phenylalanyl-tRNA synthetase (PheRS), and expresses and purifies trypanosoma brucei phenylalanyl-tRNA synthetase protein, thus laying a foundation for the screening of antiparasitic agent.

Description

technical field [0001] The invention relates to a preparation method of an enzyme in the field of biotechnology, in particular to a preparation method of Trypanosoma brucei phenylalanyl tRNA synthetase. Background technique [0002] Trypanosoma brucei is a unique parasite in sub-Saharan Africa. When it invades the host, it can cause African trypanosomiasis or sleeping sickness, causing 50,000 to 200,000 deaths per year. Trypanosomiasis is mainly transmitted to humans and livestock through the bite of tsetse flies, and the early symptoms are mainly discomfort and irregular fever; if left untreated, the parasite invades the central nervous system, eventually leading to coma and death. There are some defects in the drugs currently on the market, such as drug toxicity, lack of resistance to parenteral administration, etc., all of which need to be solved. [0003] In recent years, with the vigorous development of biochemistry and the successive deciphering of the trypanosome gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/70C12R1/19
Inventor 姚璎杲光伟李大伟
Owner 泰州新生源生物医药有限公司
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