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Method for recombining human PSP94 protein secretory expression in colibacillus

A technology of Escherichia coli, secretion expression, applied in the field of human PSP94 protein, which can solve the problems of cumbersome processing, heavy workload, and low yield of active products, and achieve the effects of avoiding degradation, promoting cell apoptosis, and easy separation and purification

Inactive Publication Date: 2005-09-28
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to overcome the cumbersome post-processing of existing PSP94 expression, heavy workload, and low yield of active products, the present invention provides a method for secreting and expressing recombinant human PSP94 protein in Escherichia coli

Method used

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  • Method for recombining human PSP94 protein secretory expression in colibacillus
  • Method for recombining human PSP94 protein secretory expression in colibacillus
  • Method for recombining human PSP94 protein secretory expression in colibacillus

Examples

Experimental program
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Effect test

Embodiment

[0042] 1. Cloning and amplification of PSP94 cDNA

[0043] The total RNA of human normal prostate tissue was extracted by guanidine isothiocyanate one-step method, cDNA was synthesized by AMV reverse transcriptase under the guidance of primer Oligo(dT)15, and PSP94 cDNA was amplified by PCR using CP1 and CP2 as primers.

[0044] CP1 (5'TTACTGATAGGCATGGCTAC-3')

[0045] CP2 (5'-TGCTTATCACAATGAATGTTCTCCTGGGG-3')

[0046] After gel recovery, the amplified product was ligated with the pUC19 plasmid digested with SmaI to construct the cloning plasmid pUC-PSP94. The ligation product was transformed into competent and E.coli JM109 bacteria, spread on the LB agar plate containing ampicillin, IPTG and X-Gal, cultured upside down at 37°C overnight, selected white colonies and inoculated in the LB liquid medium containing ampicillin, After shaking and culturing at 37°C overnight, a small amount of plasmids were extracted by alkaline lysis method, and the PSP94 protein gene fragment was a...

experiment example

[0140] Experimental example The present invention recombinant PSP94 protein activity analysis

[0141] The purified recombinant protein was quantified by Bio-Rad Protein Assay, and the concentration of PSP94 was adjusted to (1000, 500, 100, 50, 10, 5, 0) × 10 with RPMI-1640 culture medium. -4 M, filter sterilized.

[0142] (1) MTT method was used to determine the concentration of prostate cancer PC-3 cells to 2×10 5 / mL, inoculated in a 96-well flat-bottom culture plate, 100 μl per well. After culturing for 24 hours, the supernatant was discarded, and 200 μl of culture solution containing different concentrations of PSP94 was added to each well, and 3 replicate wells were set up in each group. The negative control was the purified supernatant of the periplasmic space of bacteria containing only plasmid pBV220 cultured at the same concentration at the same time. After 44 hours of cell culture, add freshly prepared 5 μg / mL MTT to each well, incubate in a 37°C incubator for 4 h...

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Abstract

The present invention relates to method of secreting and expressing recombinant human PSP94 protein in colibaccillus, and belongs to the field of the expression of bioactive human PSP94 protein. The present invention includes extracting total prostate tissue RNA, obtaining Chinese PSP94 cDNA sequence via RT-PCR process, inserting it into cloned vector pUC19, determining sequence after coarse PCR screening; connecting double restricted PSP94 protein coding gene, signal peptide and pBV220 plasmid to constitute recombinant plasmid pBV-PSP94; transforming colibaccillus strain with the positive recombinant plasmid pBV-PSP94 and protein inducing expression at optimal temperature 40 deg.c for optimal time of 5 hr. The present invention has the beneficial effect of mutating the signal peptide of secreting expression vector and inducing restriction site to the signal peptide superiorly by means of molecular biological technology.

Description

technical field [0001] The invention relates to a method for secreting and expressing biologically active human PSP94 protein by using Escherichia coli. Background technique [0002] Prostate cancer is a representative disease of older men. Since 1996, the incidence of prostate cancer in the United States has ranked first in male malignant tumors, and its mortality rate is second only to lung cancer. Because the location of the prostate is concealed, and most cancers occur in the peripheral area away from the urethra, modern detection techniques such as serum PSA detection are difficult to meet the needs of early diagnosis, and many patients lose the opportunity for treatment. [0003] So far, clinical prostate cancer in my country can only be diagnosed at an advanced stage. Since 1999, the "Prostate Disease Prevention and Research Center" where our research group is located, with the assistance of the special technical cooperation project between the Chinese and Japanese ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70
Inventor 田长生高瑞娟李扬赵丹刘喜春赵雪俭
Owner JILIN UNIV
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