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Method for producing triose phosphate isomerase

A technology of triose phosphate and isomerase, applied in the biological field, can solve the problems such as delay of effective treatment time, low detection rate, influence control, etc.

Inactive Publication Date: 2007-06-13
中国农业科学院上海家畜寄生虫病研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Pathogen inspection includes: feces washing and sedimentation method, sedimentation method and miracidium hatching method. The disadvantages of the above methods are: the detection rate is low, and only when the worms mature in the body can the eggs be detected from the feces. At this time, the acute phase has passed, and most of the harm of oriental fluke to cattle and sheep is caused by the damage caused by the migrating parenchymal organs in the acute phase, which delays the effective treatment time and affects the control of the disease
However, in the prior art, there is still no effective method for high expression of triose phosphate isomerase

Method used

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  • Method for producing triose phosphate isomerase
  • Method for producing triose phosphate isomerase
  • Method for producing triose phosphate isomerase

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Experimental program
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Effect test

Embodiment 1

[0053] Cloning of embodiment 1 triose phosphate isomerase full-length gene

[0054] 1.1 Amplification of large fragments of TPI gene

[0055] A pair of primers were designed using Dnaman according to the conserved region of the reported cDNA sequence GenBank U50847 of the triose phosphate isomerase gene of Schistosoma japonicum strain GenBank U50847 and the cDNA sequence GenBank M83294 of the triose phosphate isomerase gene of Schistosoma mansoni:

[0056] P1: 5'-TGTCTGGATCTCGGAAA-3' (SEQ ID NO: 1)

[0057] P2: 5'-GCTCCACCATAGATAATACG-3' (SEQ ID NO: 2)

[0058] Using oligo(dT)18, the cDNA sequence of TPI gene was reverse-transcribed from the total RNA of Orimilha turkestania adults. Then use P1 and P2 to amplify a large fragment of TPI gene. The PCR reaction parameters are: 94°C for 2min, 94°C for 45s, 53°C for 1min, 72°C for 1min, 35cycles, and 72°C for 10min.

[0059] 1.2 Amplification of the 5' end sequence of the TPI gene

[0060] Three primers were designed according...

Embodiment 2

[0073] The construction of embodiment 2 recombinant plasmids

[0074] 2.1 Amplification of the full-length sequence of the TPI gene

[0075] According to the registered TPI full-length cDNA sequence, design a pair of specific primers containing EcoRI and NotI restriction sites,

[0076] P1: 5'-GCAGAATTCGAGAAAATGTCTGGATC-3' (SEQ ID NO: 8)

[0077] P1: 5'-GGTGCGGCCGCGTTCGAACTAATAAGGATG-3' (SEQ ID NO: 9)

[0078] Using oligo(dT) 18 The cDNA sequence of the TPI gene was reverse transcribed from the total RNA of the adult Orchidina turkestan. Then amplify the full-length sequence of TPI through P1 and P2. The PCR parameters are: 94°C for 2min, 94°C for 45s, 52°C for 30s, 72°C for 30s, 5 cycles, 72°C for 10min. The PCR product is connected by TA cloning On the pMD18-T Vector, PCR identification is correct and then sequenced.

[0079] 2.2 Construction of pPIC9k-TPI

[0080] The recombinant vector pMD18-T / TPI and the expression vector pPIC9k (provided by the Biotechnology Center...

Embodiment 3

[0084] Transformation and screening of embodiment 3 recombinant plasmids

[0085] 3.1 Transformation of recombinant plasmids

[0086] The recombinant plasmid was digested with SalI (37°C for 5h) to make it linearized, and transformed into the prepared competent yeast cell GS115 (provided by the Biotechnology Center of Shanghai Academy of Agricultural Sciences) by electroporation at 1.5kv, 200Ω, 25μF.

[0087] 3.2 Screening of multi-copy recombinant plasmids

[0088] The bacterial solution transformed by electroporation was spread on the MD plate of histidine-deficient medium, cultured at 30°C, and colonies appeared after 3 days. Each grown colony was continuously cultured on three 96-well cell culture plates for one week to make the bacterial solution have the same cell density, and the bacterial solution was added dropwise on a YPD plate containing 1 mg / ml G418, and incubated at 30°C until the colony grew.

[0089] As a result, multi-copy plasmid strains were obtained.

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Abstract

The invention discloses a method to produce triosephosphate isomerase, which includes the following steps: (1) The full-length of triosephosphate isomerase gene was cloned into expression vector, and was amplified; (2) The vector was cutted with restricted enzyme to linearize, then was transformed into Pichia pastoris for expression triosephosphate isomerase; (3) The expression products were collected. The invention also disclosed the application of Pichia pastoris in the production of triosephosphate isomerase. This method is low cost and high yield. The expression level of triosephosphate isomerase in Pichia pastoris can reach up to 1200mg / l, which are several times higher than prokaryotic expression system and is easy for purification, and to facilitate application in practice.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the production of an enzyme. More specifically, the invention relates to a production method of triose phosphate isomerase. Background technique [0002] Oriental flukes are a kind of schistosomiasis caused by various trematodes of the genus Orientale parasitizing in the portal vein and mesenteric vein of mammals such as cattle and sheep. Chronic infection can cause anemia, diarrhea, edema, dysplasia in livestock, affect conception or miscarriage, and acute infection can cause death of livestock such as cattle and sheep. The disease is widely distributed in our country and brings great economic losses to animal husbandry. At the same time, the cercariae of oriental fluke can cause people to suffer from cercarial dermatitis, also known as rice field dermatitis, which seriously affects human health and is a very important zoonotic parasitic disease. [0003] The diagnosis of oriental fl...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/81C12N1/19C12R1/84
Inventor 何国声魏佳徐梅倩朱顺海曹杰赵其平张大兵梁婉琪黄燕严凤高兴春郭敏黄侠姚宝安
Owner 中国农业科学院上海家畜寄生虫病研究所
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