Containing anti-apoptosis gene bcl -2 recombinant retrovirus
recombinant and identification of
【Abstract】 Objective: To construct and identify the mouse bcl -2 gene-containing recombinant retrovirus. Methods: The anti-apoptosis gene bcl -2 fragment from the vector pcDNA3.1 in the cut, and directed into retroviral vector PLNCX2, the restriction enzyme digestion; liposome into the retroviral packaging cell line PT67, G418 Screening to establish stable cell lines expressing bcl -2. Results: The double-enzyme
digestion, the success of recombinant retroviral vector; recombinant retrovirus into packaging cell PT67, after G418 selection, resistant colonies formed, and measured the virus titer was 3 × 1011cfu / L, prompt construction containing
mouse bcl -2 gene recombinant retroviral vector and stable packaging cell line successfully. Conclusion: The anti-
apoptosis gene bcl -2 containing recombinant retroviral vector was successfully constructed.
Key words recombinant retroviral vector gene bcl -2
0 Introduction
With some of the common blinding eye disease prevention and control measures have been better, refractory or non-
government eye disease has become an eye research hot spot and focus. Refractory eye disease involving mainly refers to the
number of retinal degenerative diseases, such as hereditary retinal degeneration, age-related macular degeneration,
retinal pigment epithelium (RPE), and cone-rod (photoreceptor)
cells of the progressive degeneration and death, yet so far No
to prevent the degeneration and death of these cells in an effective manner. Us to build anti-apoptosis gene bcl -2
recombinant retrovirus to import it a normal SD rats were
cultured RPE cells, then micro-injected into mice with
retinitis pigmentosa subretinal space of retinal degenerative disease genes Treatment of conduct exploratory research, in order to construct and identification of bcl -2 gene
recombinant retroviral coverage of work are as follows.
1 Materials and methods
1.1 Materials PLNCX2 retroviral vectors and packaging cell line PT67 for Microbiology, Second Military Medical University, teaching and research to provide Pan Wei, containing mouse bcl -2 gene in pcDNA3.1 (restriction sites (KPn ? Xba ?)) from the Biochemistry, Chinese Academy of
cells, Professor Liu Xinyuan as gifts, plasmid extraction kit and DNA Purification kit were purchased from Bio-Technology
Co., Ltd. Shanghai gaming; TOP10F 'and DH5α strains of E.
coli provided by the micro-teaching and research in order to
CaCl2 method for producing competent cells; various restrictions on of endo-enzymes pMD18T vector Po Sang works were purchased in TaKaRa (Dalian Co., Ltd.); NIH3T3 cells, liposomes, and G418 were purchased from Clontech, Inc.; agarose and various chemical and biological agents were purchased in Shanghai Waston bio-engineering products.
1.2 Methods ? pcDNA3.1 (bcl -2) amplification: The mouse pcDNA3.1 (bcl -2) plasmid into E. coli using CaCL2 way TOP10F 'strains, LB medium was amplified alkaline lysis method of
extracting qualitative tablets DNA, and plasmid was extracted using Wizard plasmid purification kit to be DNA. ? the design
and synthesis of primers (with Hind ? and Stu ? two enzyme
restriction sites), upstream 5'-ATA AAG CTT ATG GCG CAA GCC
GGG AGA-3 '(with Hind ? restriction sites), downstream 5'-TAT
AGG CCT TCA CTT GTG GCC CAG GTA-3 '(with Stu ? restriction
site), by the Shanghai Public Health and synthesis of bio-
engineering company. (3) PCR amplification: 50μL system, the
template DNA1μL, dNTP1μL (200μmol / L), the upstream primer 5'-ATA AAG CTT ATG GCG CAA GCC GGG AGA-3 '(with Hind ?
restriction sites), downstream 5 '-TAT AGG CCT TCA CTT GTG GCC CAG GTA-3' each 1μL (25pmol), Taq Buffer5μL, Taq enzyme 1μ
L, in the PCR instrument (2400, U.S. PE Corporation) was
amplified, 94 ? 5min after 94 ? 30s, 55 ? 30s, 72 ? 50s,
35 cycles, 72 ? 7min, 4 ? 10min. PCR amplified product
sequence analysis of DNA amplified by electrophoresis, low-
melting-point plastic recycling purification. According to
Promega manual connection into pGEM-T vector, was transformed
into DH5α E. coli receptors, the selected target gene plasmid containing the bcl -2 colony, extract digested identified, named pGEM-bcl -2. Will be elected by the E. coli plasmid containing the gene delivery Shanghai GeneCore sequencing companies. With the restriction endonuclease enzyme Hind ?
and Stu ? plasmid pGEM-bcl -2, electrophoresis, low-melting-
point plastic recycling mouse bcl-2 DNA fragment (0.7kb),
dissolved in TE in, -20 ? to save. Retroviral vector PLNCX2,
with the restriction enzymes Hind ? and Stu ? digested
dephosphorylated, pGEM-bcl -2 through the same enzyme isolated 0.7bpbcl-2 after all the coding region fragments, the role of ligase in the T4DNA Next with the vector, transformation, and
using rapid screening method for screening out the gene recombinant PLNCX2-bcl -2. Transfected packaging cell line
PT67 packaging cell line recovery PT67, in complete medium (containing fetal calf serum) culture, in transfected pre-12 ~
24h, the PT67 cell line was inoculated into 60mm Petri dish, when the cell growth to 60 % ~ 80% confluence, adding a pre-
prepared mixture in the transfection of 37 ?, CO2 tank
culture incubated 24h, including recombinant retroviral vector plasmid 100μL and empty vector 100μL (both plasmid 5μg,
lipid Body 30μg, diluted with serum-free culture medium
made); the next day replacement of fresh complete medium, at 72h after transfection, aspirate medium, PBS wash cells two times and re-adding 5mL complete medium and continue to foster 48h, in order to increased virus titers. The transfected packaging cell line according to the ratio of 1:20 in the
passage containing G418 (300) the choice of culture medium 3d, the replacement for the G418 (400g / L) medium to maintain selection, to continue to foster 2wk, to be resistant colonies grow After sorting large and healthy cloned and transferred to
another culture plate each hole to join expanding culture medium without antibiotics; virus diluted accordingly, NIH3T3 cells infected with measured virus titers. Reposted elsewhere in the paper for free download http://
2 Results
Sequencing and Gene bank line to prove the success of PCR amplification with Stu ? and Hind ? restriction sites of
the two enzyme gene (Figure 1,2). PLNCX2-bcl -2 recombinant
expression vector of the restriction endonucleases shown in Figure 3. When the packaging cell growth to 60% ~ 80%
confluence, the transfection mixture by adding about a few minutes can be found in many small white particles attached to the cell surface, about 4 ~ 6h after the white particles gradually into the cell interior. When the cell culture medium
containing G418 selection, the visible cell death, as time goes by, the living shall be successfully transfected cells, the cells are colony forming gradually. Determined after amplification of the biggest viral titer 3 × 1011cfu / L.
3 Discussion
Apoptosis is energy-dependent cell death program caused
the activation of cell suicide (suicide). Therefore, from the genetic control of apoptosis, so there are known as programmed cell death, such a (procedural) cell death (programmed cell
death, PCD). Available information indicates that in terms of physiological or pathological state of the PCD, bcl -2 gene
are critical regulatory factors, bcl -2 gene was first
discovered in the No. 14 and No. 18 chromosome balance of ectopic site, and there is a in most human follicular cell lymphoma [1]. bcl -2 is a B-cell lymphoma or leukemia -2 gene
(B-cell lymphoma / leukemia-2) English acronym. Of the gene in
1985, first in the majority of non-Hodgkin's lymphoma (non-
Hondgkin B-cell lymphomas) on the result of chromosomal
translocation and activation of genes found. In 1990 revealed that the gene have a special role in inhibiting PCD. bcl -2
have such a good role in inhibiting cell apoptosis, then we are for those with apoptosis in the pathological mechanisms of
neurodegenerative diseases, it is possible to find a way out, of course, we need to find effective and safe "transport equipment" The cells will be imported to make it play a role. Has been an ongoing study of viral vectors, there are
retroviruses, adenoviruses, adeno-associated virus, and slow
virus. Bennett et al [2] used the adeno-virus bcl-2 gene into
mice and found that bcl -2 allows it to significantly slow
down the degeneration of photoreceptor cells in mice and the delay would only occur after birth 6wk. Retroviral gene transfer into cells as a means of delivery, application of the earliest research is quite mature, and are still being widely used. China has reported the successful construction of antisense human bcl -2 gene retroviral expression vector
[3,4], also reported a successful build with a complete expression of bcl -2 region recombinant expression vector, and the reading frame starting with the bcl -2 Some parts of the
coding region of the pros and cons to the recombinant
retroviral expression vector [5]. This PLNCX2-bcl -2 was
successfully constructed recombinant expression vector. For the latter part of retinitis pigmentosa of research and experiments laid a theoretical basis and data for reference.
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results in a delay in photoreceptor cell death in the rd / rd mouse. Gene Ther, 1998; 5:1156-1164
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