Medicine for curing diarrhea and its preparing process

A technology of drugs and compositions, applied in the fields of biotechnology and medicine, which can solve the problems of low ITF content, difficulty in structure and function research, etc.

Inactive Publication Date: 2008-01-09
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the extremely low content of ITF in tissues, it is very difficult to directly isolate native ITF for structural and functional studies

Method used

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  • Medicine for curing diarrhea and its preparing process
  • Medicine for curing diarrhea and its preparing process
  • Medicine for curing diarrhea and its preparing process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Embodiment 1 plasmid construction

[0112] Plasmids and strains: Plasmid pPIC9 is a product of Invitrogen; strain TG1 is a product of Sigma.

[0113] Enzymes and reagents: Restriction enzymes (XhoI, NotI) and T4 DNA ligase are products of TaKaRa; total cell RNA extraction kit Trizol, reverse transcription reagents and PCR reagents are all products of GIBCO BRL; DNA gel The recovery kit and plasmid purification kit were products of Shanghai Huashun Bioengineering Co., Ltd.; pfu polymerase was purchased from Shanghai Sangon Bioengineering Company; primers were synthesized and purified by Shanghai Sangon Bioengineering Company; DNA Marker was GeneRuler 100bp DNA Ladder and DL 2000. Other reagents were imported or domestic analytically pure.

[0114] 1. Extraction of total RNA from human colonic mucosa

[0115] The intestinal mucosa was biopsied by colonoscopy and immediately put into liquid nitrogen for storage at -80°C. Colonic mucosa was placed in a homogenizer, and ...

Embodiment 2

[0133] Expression and purification of embodiment 2 protein

[0134] 1. Recombinant plasmid transformation

[0135] Extract about 10ug of the recombinant plasmid, digest it with SalI, ethanol precipitate and recover the linearized plasmid, and transform it into GS115 (Mut + his4) In yeast competent cells, the electric shock products were spread on MD plate (13.4g / LYNB, 20g / L glucose, 4×10 -4 g / L biotin, 15g / L agar), cultured at 30°C for 2-3 days, and observed the growth of transformants.

[0136] 2. Identification and screening of transformants

[0137] According to the operation manual provided by Invitrogen, select universal primers 5'AOX1 and 3'AOX1, and amplify the target fragment by colony PCR to screen positive transformants. Add 30 cycles.

[0138] 5'AOX1: 5'-GACTGGTTCCAATTGACAAGC-3' (SEQ ID NO: 3);

[0139] 3'AOX1: 5'-GCAAATGGCATTCTGACATCC-3' (SEQ ID NO: 4).

[0140] Pick a single colony from the MD plate into the YPD medium (10g / L yeast extract, 20g / L peptone, 1...

Embodiment 3

[0179] Example 3 Identification of biological activity of hITF

[0180] 6.1 Method

[0181] Rat small intestinal epithelial cells (IEC-6, purchased from the Cell Collection Center of Peking Union Medical College, China) and a cell migration kit (Corning, US) were used to observe the effect of different concentrations of recombinant ITF on cell migration. Cells were cultured in serum-free medium for 24 hours to cause cell starvation, and then digested with trypsin to prepare cell suspension with a cell concentration of 0.5-1×10 6 / ml, add 300ul of cell suspension to the end of each built-in well plate of the 24-well plate (the bottom is a membrane with a pore size of 0.8um, and epithelial cells can pass through the well), and add physiological saline or 10 -9 M, 10 -7 M and 10 -5 For the recombinant ITF of M, add 500ul medium containing 5% fetal bovine serum to the outside of the built-in well plate. After culturing for 14 hours, suck out the cell suspension in the built-in ...

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Abstract

Process is concerned with high active human enteric trilobite factor protein and product concerned. Also, its usage in preparation of pharmaceutics is disclosed herewith. Meanwhile, ITF protein with high ratio of double bodies is obtained with low cost.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and in particular relates to a medicine for treating diarrhea and a preparation method thereof. Background technique [0002] Diarrheal disease is a common and frequently-occurring disease in pediatric clinical medicine. According to the World Health Organization, about 1 billion people suffer from diarrhea every year, 500 million of which occur in the third world. 4-5 million children die each year. According to the 1988 survey data of the China Epidemic Prevention Department, there are estimated to be about 863 million cases of diarrhea (including adults and children) in China every year, and the average incidence rate of children under 5 years old is 2.5 times per person. It is estimated that there are 294.5 million cases of diarrhea in children under 5 years old every year Diarrhea, it can be seen that the harm is great. Although there are many kinds of drugs for the treatment of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02A61K38/17A61P1/04A61P1/12
Inventor 许春娣杨晟刘伟钟雪梅周莎
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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