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Lentiviral plasmid expression vector as well as construction method and application of lentiviral plasmid expression vector

An expression vector and plasmid vector technology, applied in the field of lentiviral plasmid expression vector and its construction, can solve the problems of low sensitivity and specificity, increased human error, increased environmental hazards and the like

Inactive Publication Date: 2014-09-03
GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ELISA is a commonly used method, but its sensitivity and specificity are not high; RIPA has high sensitivity and specificity, but its application is limited due to the adverse effects of radioactive substances on the environment; currently, the sensitivity and specificity of CBA and FIPA Ideal for specificity
However, the average detection period of the CBA method exceeds two days, the fluorescence efficiency is unstable, and human observation is required, which is time-consuming and laborious, and may increase human errors in the detection; the FIPA method also involves the application of isotopes, and the environmental hazards are greatly increased.
In addition, the detection of MuSK autoantibodies currently only a small number of researchers in China have done scientific research using CBA and FIPA, and they have not been routinely used in clinical practice. Therefore, which method is recommended as the "gold standard" for clinical MuSK antibody detection remains to be clarified

Method used

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  • Lentiviral plasmid expression vector as well as construction method and application of lentiviral plasmid expression vector
  • Lentiviral plasmid expression vector as well as construction method and application of lentiviral plasmid expression vector
  • Lentiviral plasmid expression vector as well as construction method and application of lentiviral plasmid expression vector

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Experimental program
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Embodiment 1

[0039] A lentiviral plasmid expression vector is pCDH-MCS-MuSK-GFP, its nucleotide sequence is shown in the sequence listing as SEQ ID NO.2, and its construction method is carried out according to the following steps:

[0040] 1) Vector construction: using human muscle tissue cDNA as a template, using primers MuskF: 5'-CCAGCTAGCATGAGAGAGCTCGTCAACATTCCAC-3' and MuskR 5'-CACAGCGGCCGCTTAGACACTCACAGTTCCCTCTGCC-3' to amplify the MuSK isoform 2 cDNA sequence, as shown in SEQ ID NO.1 in the sequence table The specific steps are as follows:

[0041] Add 300 ng of DNA template, 0.4 μmol / L final concentration of each primer, 0.2 μmol / L final concentration of dNTP, 5 μl 10×PCR buffer, and 0.5 μl Taq DNA polymerase to the PCR reaction tube, with a final concentration of about 5 U / L. μl, ddH 2 Make up O to a final volume of 50 μl; PCR cycle program is: denaturation at 94°C for 4 minutes, 98°C for 10 seconds, annealing at 68°C for 45 seconds, extension at 72°C for 2 minutes, and a total of...

Embodiment 2

[0045] The application of constructing the detection of anti-muscle-specific receptor tyrosine kinase antibody is composed of the following steps:

[0046] 1) Using CaCl 2 The lentivirus expressing the MuSK-GFP (pCDH-MCS-Musk-EF1-copGFP) fusion protein was packaged by the method, and the virus suspension was collected to infect HEK293 cells in the presence of 6 μg / ml Polybrene. After 1 week, the sorting positive was stable transfected cells. Continue to subculture and carry out secondary screening to obtain 100% transfected HEK293 cell line stably expressing the fusion protein. Culture conditions: DMEM medium containing 10% fetal bovine serum at 37°C for transfected HEK293 stably expressing the fusion protein cell;

[0047] Specific steps: When the HEK293 cells grow to 70% density, remove the old medium, wash the cells with PBS, use the CaCl2 method to package the lentivirus expressing the MuSK-GFP fusion protein, and collect the virus suspension in 6 μg / ml Polybrene Infect...

Embodiment 3

[0055] Actual application:

[0056] The present invention randomly selects 127 AchR-positive myasthenia gravis patients, aged 13-80 years, 30 males and 37 females; 103 AchR-negative myasthenia gravis patients, aged 13-80 years, 56 males and 71 females. From 2008 to 2013, patients with myasthenia gravis who were diagnosed in the neurology clinic and ward of Tianjin Medical University General Hospital. The most important diagnostic basis for myasthenia gravis is the patient's past history, clinical presentation, and examination findings of fluctuating fatigability, especially with involvement of the extraocular and bulbar muscles. Our included patients all presented with pathological fluctuating muscle fatigue and met at least one of the following diagnostic criteria:

[0057] 1) Anticholinesterase drug—Neostigmine test leads to obvious improvement of muscle strength;

[0058] 2) Low-frequency nerve repetitive electrical stimulation motor amplitude attenuation > 15%;

[0059]...

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Abstract

The invention discloses a lentiviral plasmid expression vector as well as a construction method and an application of the lentiviral plasmid expression vector. The lentiviral plasmid expression vector is pCDH-MCS-MuSK-GFP and the nucleotide sequence of the lentiviral plasmid expression vector is shown in SEQ ID NO. 2 in a sequence table. The invention further discloses an application of the lentiviral plasmid expression vector in construction of an anti-muscle-specific receptor tyrosine kinase antibody detection. According to the invention, a stable transfected cell line of MuSK is established by constructing a viral vector, a MuSK antibody is qualitatively and quantitatively detected clinically by an immunofluorescence staining method with relatively high sensitivity and specificity in combination with a flow cytometry, the detection results are more stable and manpower and time costs are greatly saved. The diagnosis and treatment levels of myasthenia gravis are greatly improved by the establishment of the project, the labor capacity and life quality of patients are improved and the lentiviral plasmid expression vector has significant social and economic benefits.

Description

technical field [0001] The present invention relates to a lentiviral plasmid expression vector and its construction method and application, in particular to a lentiviral plasmid expression vector and its construction method and the construction of anti-muscle specific receptor tyrosine kinase (muscle specific kinase, MuSK) antibody detection Applications. Background technique [0002] Myasthenia gravis (MG) is an antibody-mediated autoimmune disease that mainly involves acetylcholine receptors (acytylcholine receptors, AchR) in the post-synaptic neuromuscular junction, leading to neuromuscular junction transmission disorders. Its clinical features are partial or general skeletal muscle fatigue, showing fluctuating muscle weakness, which is aggravated after activities, relieved after rest, and light in the morning and heavy in the evening. With the continuous improvement of the level of medical diagnosis and the change of people's living environment and other factors, the in...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66G01N33/53
Inventor 闫亚平施福东李敏淑金薇娜齐媛
Owner GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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