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Prostate gland specificity antigen diagnosis reagent kit

A prostate-specific, kit-based technology, applied in the biological field, can solve the problems of protein lack of spatial conformation, glycosylation modification, biological activity affecting the growth state and expression of recombinant cells, etc.

Inactive Publication Date: 2008-12-17
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is still difficult to use genetic engineering technology to express a large amount of recombinant PSA protein with the same or similar activity as the natural PSA protein in this field. This may be due to the fact that PSA is a functional protein, and its biological activity affects the recombinant cells. Growth status and expression
In addition, the system usually used to prepare recombinant proteins is a prokaryotic expression system. Proteins expressed in prokaryotic systems often lack good spatial conformation and basic glycosylation modifications, which are far from natural proteins.

Method used

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  • Prostate gland specificity antigen diagnosis reagent kit
  • Prostate gland specificity antigen diagnosis reagent kit
  • Prostate gland specificity antigen diagnosis reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] The gene cloning of embodiment 1.PSA

[0159] 1. Cultivation, RNA extraction and reverse transcription of CRL-1740 prostate cancer cell line

[0160] Take the CRL-1740 prostate cancer cell line (purchased from ATCC) out of liquid nitrogen, put it in sterile water at 37°C, add 10ml of RPMI1640 complete medium after the frozen cells are thawed, blow evenly, centrifuge at 1000rpm, and wash again Cells were divided into 2 × 10 6 / ml cultured at 75cm 2 T FLASK, cultured at 37°C for 2 passages.

[0161] Cell cultures yielded 5 x 10 6 For each cell, RNA was extracted by conventional methods, keeping as low temperature and RNAse-free as possible during extraction. The results of electrophoresis after RNA extraction were as follows: figure 2 As shown, RNA was extracted.

[0162] The reverse transcription system (40ul) is as follows: 5× buffer 8ul, 0.1M DTT 4ul, dNTPs (10mM) 8ul, RNAse inhibitor 0.5ul, Superscriptase 1ul, Oligo (dT) 12-16 (0.5ug / ul) 1ul, Sample RNA (4ug) ...

Embodiment 2

[0176] Example 2. Construction of recombinant plasmids

[0177] The present inventors cloned the signal peptide of the AcNPV nuclear polyhedrosis virus (AcNPV) membrane protein GP64, the specific sequence is as follows: atgctactagtaaatcagtcacaccaaggcttcaataaggaacacacaagcaagatggtaagcgctattgttttatgtgcttttggcggcggcggcgcattctgcctttgcg (SEQ ID NO: 17), and introduced the BamHI enzyme cutting site and Coptttgcg.

[0178] Table 2

[0179] GP64 Template-1

atgctactagtaaatcagtcacaccaaggcttcaataaggaacacaca

SEQ ID NO: 10

GP64 Template-2

ataaaacaatagcgcttaccatcttgcttgtgtgttcct

SEQ ID NO: 11

GP64 Template-3

attgttttatgtgcttttggcggcggcggcgcattctgcctttgcg

SEQ ID NO: 12

Gp64+ forward primer (Sp)

ataagaatcggtccgaaaccatgctactagtaaatcag

SEQ ID NO: 20

Gp64+ reverse primer (As)

gggatcccgcaaaggcagaatgcgc

SEQ ID NO: 21

[0180] Using Gp64+S p and Gp64+As in Table 2 as primers and GP64 templates-1, 2, and 3 as template...

Embodiment 3

[0187] Example 3. Obtaining and protein expression and purification of baculovirus

[0188] The pFastBac-GP64-PSA-Fc plasmid prepared above was transformed into DH10Bac genetically engineered bacteria, and positive clones were obtained by blue-white screening (white spot selection).

[0189] Extract the recombinant Bacmid bacmid (which carries the GP64-PSA-Fc coding gene) from the positive bacillus, use the Bacmid bacmid as a template, and perform PCR amplification with the sequences shown in Table 4 as primers to obtain amplification The product was verified by electrophoresis. Electrophoresis results such as Figure 10 .

[0190] Table 4

[0191] M13 Forward

GTTTCCCAGTCACGAC

SEQ ID NO: 15

M13 Reverse

CAGGAAACAGCTATGAC

SEQ ID NO: 16

[0192] Take the sf9 cell line out of liquid nitrogen and put it in sterile water at 27°C. After the frozen cells are thawed, add them to 10ml of SFM medium. 6 / ml cultured at 75cm 2 T FLASK, cultured ...

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Abstract

The invention belongs to the biotechnology field, discloses fusion protein of a prostate specific antigen (PSA) and G-type immunoglobulin domain (IgG Fc), and also discloses a PSA detection kit containing the fusion protein as the standard sample. The invention carries out the fusion expression of the IgG Fc and the PSA, therefore, not only a large amount of fusion protein can be obtained, but also the purification of the fusion protein is very simple, and the bioactivity of the fusion protein is closer to that of the natural PSA. The invention overcomes the technical defects of the difficult recombinant expression and the recombined PSA closer to the natural PSA in the prior art, and greatly reduces the manufacturing cost of the PSA detection kit.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, the invention relates to a specific detection kit for prostate cancer. Background technique [0002] Prostate cancer is a common and frequently-occurring disease in middle-aged and elderly men. According to statistics, among male cancer patients, prostate cancer has the highest incidence rate and is the third leading cause of death after lung cancer and colorectal cancer. Prostate-specific antigen (PSA) is a glycoprotein with a molecular weight of 34KD. It has a high degree of tissue specificity and is distributed in normal prostate tissue, prostatic hyperplasia tissue, prostate malignant tissue, metastatic cancer, prostate secretion fluid, and seminal plasma. PSA is currently the most important and widely used prostate cancer marker in the diagnosis of prostate cancer. Measuring the prostate specific antigen (PSA) in serum can be used for screening and early diagnosis of prostate...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N33/566
Inventor 孙兵袁建伟黄超峰胡宇蔡兴峰
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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