Echinococcus granulosus developmental-stage secretory protein expression gene chip

A technology of Echinococcus granulosus and gene chip, which is applied in protein nucleotide library, microbial determination/inspection, chemical library, etc. Effect

Active Publication Date: 2013-02-27
THE FIRST TEACHING HOSPITAL OF XINJIANG MEDICAL UNIVERCITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, for organisms that have not undergone whole-genome sequencing, such as the...

Method used

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  • Echinococcus granulosus developmental-stage secretory protein expression gene chip
  • Echinococcus granulosus developmental-stage secretory protein expression gene chip
  • Echinococcus granulosus developmental-stage secretory protein expression gene chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the construction of cDNA library

[0028] The Creator SMART cDNA library construction kit provided by Clontech was used to construct the Eg cDNA library according to its instructions.

[0029] 1. Extraction of total RNA

[0030] The method of Chomezynski and Sacchi (1988) was used for preparation of total RNA and oligo-dT cellulose chromatography for poly(A)+ RNA (Sambrook et al., 1989).

[0031] Protoscoleum of Echinococcus granulosus was extracted from fresh goat liver tissue suffering from cystic echinococcosis, and frozen in liquid nitrogen. Take out 100 mg of tissue, grind it into powder in liquid nitrogen, add 1 ml Trizol solution for grinding, place the grinding solution at room temperature for 5 minutes, transfer it to a 2 mL glass tube (treated with DEPC), homogenize it on ice for 20 times, and transfer it to 1.5 mL EP tube, then add 0.2ml of chloroform per 1ml of Trizol solution, cap the centrifuge tube tightly, shake the centrifuge tube vigorous...

Embodiment 2

[0063] Example 2, cDNA library sequencing

[0064] 1. Colony PCR identification

[0065] (1) Randomly select 40,000 single colonies from the cDNA library and culture them overnight in 5mL LB medium. Take 1 μl of bacterial solution from each tube as a template, and perform PCR amplification with M13 universal primers (forward primer is 5'-CCCAGTCACGACGTTGTAAAACG-3', reverse primer is 5'-ACGGATAATTTCACACAGG-3') to confirm Whether the clone has the target fragment inserted and whether it is a single product.

[0066] (2) Establish the PCR reaction mixture (MasterMix) according to the conditions in Table 2 (that is, the PCR reaction formula table in Table 2)

[0067] (3) Mix well and centrifuge briefly.

[0068] (4) Carry out PCR reaction, the reaction parameters are as follows: 94°C for 30 seconds for 1 cycle, (94°C for 30 seconds, 68°C for 3 minutes) for 35 cycles, 68°C for 5 minutes for 1 cycle.

[0069] (5) At the end of the cycle, take 5ul products respectively, and obser...

Embodiment 3

[0074] Example 3 Preparation of gene chip

[0075] 1. Re-amplification of new expressed sequence tags

[0076] Select the PCR products of SEQ ID No.1 to SEQ ID No.30 as templates, dilute them 100 times, and carry out PCR amplification (DNAengine PCR instrument-Bio-Rad, USA) respectively. The primer is the universal primer M13 (the forward primer is 5'- CCCAGTCACGACGTTGTAAAACG-3', the reverse primer is 5'-AGCGGATAATTTCACACAGG-3'), each 100 μl reaction system includes: 10×PCR buffer 10 μl; 25 mmol / L MgCl 2 7 μl; 1 μl each of forward and reverse primers of 100 ng / μl, 1 μl of 20 mmol / L dNTP, 3 U of Tag DNA Polymerase (Promega, USA) and about 10 ng of plasmid template. PCR amplification process: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s in each cycle, annealing at 58°C for 60 s, extension at 72°C for 120 s; after 36-40 cycles, extension at 72°C for 10 min.

[0077] 2. PCR product processing

[0078] Add 200 μl (2 times the volume) of ethanol or 100 μl of...

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Abstract

The invention discloses an echinococcus granulosus developmental-stage secretory protein expression gene chip. The echinococcus granulosus developmental-stage secretory protein expression gene chip is characterized in that sequences of isolated cDNA expression sequence labels of echinococcus granulosus protoscolex, vesicle, oncosphere and adult developmental stage secretory proteins are combined with vectors; and the cDNA expression sequence labels are shown in the formulas of SEQ ID No.1 to SEQ ID No.30. The echinococcus granulosus developmental-stage secretory protein expression gene chip has commercial values and scientific research values. Through the novel cDNA expression sequence labels, a genetic map is drawn. The echinococcus granulosus developmental-stage secretory protein expression gene chip has an important effect of research of secretory protein gene expression. The expression sequence labels can realize screening of targets of a cystic echinococcosis-resistant drug and through the expression sequence labels, the optimal echinococcosis-resistant drug molecules are selected out. The echinococcus granulosus developmental-stage secretory protein expression gene chip can be used for screening of a cystic echinococcosis diagnostic marker and can be used for research and development of an echinococcosis diagnostic kit having high specificity. The echinococcus granulosus developmental-stage secretory protein expression gene chip can be used for screening of cystic echinococcosis protective antigens and can be used for research and development of echinococcosis vaccines suitable for the animal husbandry and pet dogs.

Description

technical field [0001] The invention relates to the technical field of biochips, and is a gene chip composed of cDNA expression sequence tags expressing secreted proteins in the developmental stage of Echinococcus granulosus, and especially relates to protoscolums, vesicles, hexacarpas, and adults of Echinococcus granulosus Four developmental stages expressing cDNA expression sequence tags of secreted proteins and their constituent gene chips. Background technique [0002] Transcribed and expressable sequences (genes) in biological genomes only account for 3-5% of the total sequences. Determination of this part of the sequence will directly lead to the discovery of new genes and obtain the information most closely related to industrialization in the genome. The whole genome size of Eg is about 150Mb, and the predicted number of genes is about 10,000, which is less than that of Schistosoma japonicum (15,000). So far, only a few genes have been obtained, and the understanding...

Claims

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Application Information

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IPC IPC(8): C40B40/08C12Q1/68
Inventor 温浩吕国栋张文宝林仁勇卢晓梅王俊华张传山
Owner THE FIRST TEACHING HOSPITAL OF XINJIANG MEDICAL UNIVERCITY
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