Novel HER2/neu gene-modified dendritic cell vaccine

A technology of dendritic cells and genetic modification, applied in the fields of biology and medicine, can solve the problems of short expression time, insufficient persistence and strength of immune effect, weakening DC stimulation of immunity, etc.

Active Publication Date: 2011-02-09
CHANGSHA HUI LIN LIFE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the three gene transfer methods mentioned above can produce effective DC HER2 Vaccines, but each has some shortcomings: the expression time of the transgene after adenovirus infection of DC is relatively short, which may lead to insufficient persistence and intensity of the immune effect; retrovirus can only infect cells in the dividing stage, and clinically more The differentiation of patients' peripheral blood mononuclear cells into DCs is rarely accompanied by cell proliferation, which partially limits its possible clinical application; mRNA electroporation also has the problem of short half-life of antigen presentation. In addition, there are studies Shows that mRNA electroporation impairs DC's ability to stimulate immunity
In addition, an important feature of recombinant lentivirus infection is the long-term expression of the transgene, which may stimulate the immune system to produce a durable and enhanced immune response for DC, but on the other hand may also lead to immune tolerance, that is, using The vaccine it makes may not be effective
Currently, recombinant lentiviruses are not used to generate DC HER2 vaccine coverage

Method used

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  • Novel HER2/neu gene-modified dendritic cell vaccine
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  • Novel HER2/neu gene-modified dendritic cell vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of recombinant lentiviral vectors carrying HER2 / neu gene fragments

[0027] 1. PCR amplification and purification of Neu gene fragment

[0028] Primer sequence upstream 5'-ggaattccagcctggtccagcctgag-3'; downstream 5'-gactagttcactgtctccttcgtttgatt-3'. The reaction template is the plasmid pMMTV-neu carrying the full-length cDNA of neu, which is constructed in this unit according to the method in the prior art. The amplification reaction used the Phusion ultra-fidelity PCR kit from NEB Company. The reaction system is as follows (volume unit is μl):

[0029]

[0030]

[0031] A total of two tubes were amplified. The program is set as follows: 98 degrees for 30s→95 degrees for 10s→62 degrees for 30s→72 degrees for 40s→72 degrees for 5min→4 degrees, repeat 30 cycles. PCR instrument is the product of eppendorf company.

[0032] The PCR product was run on a gel, and the product was purified with a gel recovery and purification kit from TaKaRa C...

Embodiment 2

[0046] React at 37 degrees for 30 minutes. For the results of rubber running identification, see figure 2 . The recombinant vector was named pSin-EF2-neu-Pur. Example 2 Packaging Preparation and Concentration of Recombinant Lentivirus (rLVneu) Carrying HER2 / neu Gene Fragment

[0047] 1. Preparation of Plasmids

[0048] Including pSin-EF2-neuET-Pur, pCMV8.91 and pMD.G three kinds of plasmids. After large-scale culture of the bacteria containing the plasmid, the plasmid was prepared using the Invitrogen company's large-scale extraction kit.

[0049] 2. Preparation of transfection reagent

[0050] 0.3M CaCl 2 : 11.025g CaCl 2 2H 2 Dissolve O into 240ml Milli-Q water, dilute to 250ml, sterilize by 0.22μm filter, and store at 4°C.

[0051] 2×HBS : Add 14ml 5M NaCl solution, 12.5ml 1M HEPES buffer and 250μl 1.5M NaCl to 200ml Milli-Q water 2 HPO 4 Solution, adjust the pH value to 7.10 with NaOH or HCl, dilute to 250ml, sterilize by 0.22μm filter, and store at 4°C. ...

Embodiment 3

[0071] Example 3 Using rLVneu to infect mouse bone marrow-derived dendritic cells to prepare new DCs HER2 Vaccine (culture scale for 1 6-well plate)

[0072]The complete medium of DC is RPMI1640 medium containing 10% FBS, 2mM L-glutamine, 50μm β-mercaptoethanol and 20ng / ml GM-CSF. GM-CSF is a powder. After rehydration, it can be divided into 100ng / μl, 10μl / tube and frozen at -20 degrees. Avoid repeated freezing and thawing, and add it before use. The medium added with GM-CSF should be stored at 4°C and must be used within 7 days.

[0073] Except for separating the bones, the rest were strictly aseptic. Bacteriological or tissue culture-untreated dishes / plates are best used for DC culture.

[0074] (1) Take the femur and tibia of the mouse, soak them in alcohol for disinfection, and bring them into the ultra-clean bench for operation.

[0075] (2) Soak and wash the bones in RPMI1640, cut off the epiphysis, aspirate 10ml of RPMI1640 with a syringe, and use a 0.45mm injection...

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Abstract

The invention provides a preparation scheme of a novel HER2/neu gene-modified dendritic cell vaccine, which comprises: 1, constructing a lentivirus vector carrying the HER2/neu gene fragment; 2, preparing recombinant lentiviruses carrying the HER2/neu gene fragment by using the vector; and 3, infecting dendritic cells with the recombinant lentiviruses and obtaining the vaccine. Animal experiment proves the novel HER2/neu gene-modified dendritic cell vaccine can effectively prevent and treat HER2/neu positive tumors.

Description

technical field [0001] The invention relates to the fields of biology and medicine, and more specifically relates to the preparation of a novel HER2 / neu gene-modified dendritic cell vaccine. The invention can be applied to the prevention and treatment of HER2 / neu positive tumors. Background technique [0002] HER2 / neu is an oncogene that belongs to the epidermal growth factor receptor family. The overexpression of HER2 / neu gene frequently occurs in breast cancer, ovarian cancer, endometrial cancer, lung cancer and gastrointestinal tract tumors, and its overexpression in breast cancer is a related factor of poor prognosis. Herceptin, a monoclonal antibody against HER2 / neu, can effectively reduce the recurrence risk of HER2-positive breast cancer. The latest research shows that the overexpression of HER2 / neu can promote the proliferation of tumor stem cells and enhance their invasiveness. The above facts show that HER2 / neu is an ideal target for the treatment of HER2 / neu po...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P35/00C12N15/86C12N15/11A61K39/00
Inventor 卢光琇陈濂生卢光莹李工博
Owner CHANGSHA HUI LIN LIFE TECH
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