CN1127570C - Application of castor silkworm cell nucleus and skeleton combination elements - Google Patents

Abstract

A castor silkworm SAR element is about 1,000bp in length and can specifically combine with the nucleus skeleton. Apply the SAR element to the construction of a eukaryotic gene expression vector, that is, insert the SAR into the plasmid pLu to construct a pLu-SAR eukaryotic gene expression vector, transfect NIH3T3 cells, and make the exogenous gene luciferase gene in eukaryotic The expression activity in the cells was seventeen times higher than that of the control. SAR is a strong enhancer element. The use of SAR to construct eukaryotic gene expression vectors has the advantage of stably and efficiently expressing foreign genes, and can be further applied to the construction of vectors for genetic engineering and gene therapy.

Description

The application of castor silkworm cell nucleus and skeleton combination elements

The invention belongs to molecular cytobiology, eukaryotic gene expression control technique research field.Be specifically related to a novel transcriptional enhancer element---the cell nucleus and skeleton combination elements (SAR) of Semen Ricini silkworm rRNA gene, this SAR element application can improve efficient, the stably express of foreign gene in eukaryotic cell in the structure of expression vector.

Eukaryotic nucleus is after removing dissociative DNA, histone and other soluble proteins behind the nuclease digestion, a residual network-like structure is called nuclear skeleton or nuclear matrix.Nuclear skeleton is kept nuclear form, and form three-dimensional space with thousands of genes in the nucleus duplicate, transcribe, process and vital movement such as transportation is organized in certain space.On the chromatin some specific DNA zones can with the nuclear skeleton specific combination, be called nuclear skeleton land (SAR) or nuclear matrix land (MAR).SAR/MAR is usually located at the base portion of chromatin ring, determines chromatinic higher structure, and gene duplicated and transcribed regulating and controlling effect.

External detection and characteristic (the Mirkovitch J thereof that has reported histone gene bunch SAR the earliest by Mirkovitch J etc., et al., Cell, 1984,39:223～232), find that SAR is the dna sequence dna (A+T＞70%) that is rich in A, T, be about 400～1000bp, can be specifically and the nuclear skeleton protein binding.Then Cockerill and Garrard reported the immunoglobulin (Ig) kappa gene MAR (Cell, 1986,44:273-282), find that this MAR contains the recognition site of topoisomerase II; Along with the fundamental research to SAR/MAR deepens continuously, the SAR/MAR of increasing gene is detected, the function of SAR/MAR causes people's extensive interest, and Phi-VanL etc. have reported that in 90 years lysozyme of chicken gene 5 ' MAR can strengthen the transcriptional activity of allogeneic promoter.The SAR element of Semen Ricini silkworm rRNA gene cluster has been reported in our laboratory recently, and (1998,28 (1): 42～49), this element is by our clone, order-checking and identify for Zhao Mujun etc., Chinese science (C collects).The present invention is in the structure of expression vector, to improve foreign gene stability and high efficiency expression in eukaryotic cell with this SAR element application.

For this reason, the objective of the invention is the SAR element by the clone Semen Ricini silkworm, the SAR element application at the external eukaryotic gene expression vector of setting up into, is carried the foreign gene transfecting eukaryotic cells by this carrier, the stability and high efficiency that strengthens foreign gene is expressed.

Construction process about Semen Ricini silkworm SAR element clone is by at first detecting and identify the nuclear skeleton land (SAR) of Semen Ricini silkworm rRNA gene, then this SAR is inserted the EcoRI-SacII site of pBluescript (pSK+) plasmid, be built into pSK (+)-SAR and clone (see figure 1).This SAR is about 1, the dna fragmentation of 000bp, behind dna sequence analysis, show, this SAR is rich in A, T base (A+T＞70%), contain the topII site and zymic autonomously replicating sequence (ACS) waits other tumor-necrosis factor glycoproteins and structure fancy, the sequence of DNA is formed and is shown that this SAR is an important function element (see figure 2).

It is example that the present invention is set up into eukaryotic gene expression vector pLu-SAR with the SAR element, and structure is seen Fig. 3.This carrier contains SAR and reporter gene luciferin enzyme (Luciferase) gene and some selection markers.This construction of carrier is with EcoRI and SacII double digestion pSK-SAR plasmid (see figure 1), the SAR fragment is separated, mended with the Klenow enzyme then and put down, connect the BamHI joint, insert the BamHI site of pLu plasmid, be built into pLu-SAR expression vector (Fig. 3).The pLu plasmid contains SV40 promotor and luciferin enzyme gene (Luc), contains cyanomycin resistant gene (Amp ^R) and neomycin resistance gene (Neo ^R) selection markers.Luciferase gene is the foreign gene that is inserted on the plasmid vector, and it is imported eukaryotic cell, by detecting the vigor of luciferin enzyme, determines the expression of exogenous gene height.Fig. 4 is the detected result of luciferin enzyme activity.With pLu plasmid transfection cells of mamma animals NIH3T3, luciferin enzyme expression of gene is very low, with it in contrast, with pLu-SAR transfection cells of mamma animals NIH3T3, finds that SAR can strengthen about 17 times of the stably express of luciferin enzyme gene in eukaryotic cell.PAGLu as positive control (pAGLu and pLu plasmid are so kind as to give by U.S. Kohwi-Shigematsu T. laboratory), behind the rotaring copolymering NIH 3 T 3 cell, is strengthened seven times of luciferin enzyme gene expressions, similar (Bode J, et al, Science to reported in literature, 1992,255:195～197).Therefore we clone's SAR element application as a kind of enhancer element, improves about 17 times of expression of exogenous gene in carrier for expression of eukaryon, has very high activity.

The invention has the advantages that of the expression of SAR element at the horizontal regulatory gene of chromatin Structure, the carrier for expression of eukaryon pLu-SAR that utilizes the SAR element to make up, foreign gene is imported eukaryotic cell and be incorporated on the karyomit(e), SAR can strengthen the promotor vigor on karyomit(e), and genetic expression is strengthened.Because exogenous origin gene integrator is on karyomit(e), be assigned in offspring's daughter cell with cell fission, be difficult for losing, so the ability of SAR reinforcing gene expression has the advantage of stability and high efficiency, be specially adapted to gene therapy, to improve the expression of therapeutic gene.Because SAR/MAR can be combined on the nuclear skeleton, helps improving the level and the stability thereof of expressing protein, is applicable to eukaryotic cell producer gene engineering product, improves the quality.

The present invention is further elaborated by the following drawings and embodiment, but does not limit the scope of the invention.

Description of drawings:

Fig. 1 is pSK (+)-SAR clone of Semen Ricini silkworm SAR element

Fig. 2 is that the dna sequence dna of Semen Ricini silkworm SAR element is formed

Fig. 3 is the collection of illustrative plates of eukaryotic gene expression vector pLu-SAR

Fig. 4 is the detected result that Semen Ricini silkworm SAR element strengthens the relative vigor of luciferin enzyme

The detection and the clone of embodiment 1 Semen Ricini silkworm SAR element

A. separate and prepare ricinus silk gland tissue nucleus

Get 1～3 day ricinus silk gland tissue, 3～5g in 5 ages, homogenate is also filtered, homogenate buffer is an Al (0.25mol/L sucrose, 10mmol/L Tris-HCl (pH8.0), the 5mmol/L magnesium chloride), 1, centrifugal 10 minutes of 500rpm, collecting precipitation also suspends with Al, adds final concentration and be 0.5% Triton X-100, and ice bath is after 10 minutes, 1, centrifugal 10 minutes of 500rpm with Al suspension precipitation, makes the 2.2mol/L sucrose density gradient ultra-centrifugation, super centrifugal condition is 20,000rpm, 60 minutes, throw out was a nucleus.

B. the preparation of cell nucleus and skeleton

Nucleus through super centrifugation, with diiodosalicylic acid lithium salts (LIS) buffer B (5mmol/L Tris-HCl pH7.4,20mmol/L Repone K, 0.125mmol/L spermidine, 0.05mmol/L spermine, (the import packing of 0.1% digitonin, agent delivery station, Shanghai), 0.1mmol/L PMSF) suspend, then with reference to Mirkovitch (Cell, 1984,39:223～232) method prepares nuclear skeleton, is that 20mmol/L diiodo-salicylic acid lithium salts extracts histone and other soluble proteins in the nuclear with final concentration promptly, with 1,000u/mL restriction enzyme EcoRI and SacII digestion chromatin, centrifugation and nuclear skeleton bonded DNA.

C. the detection of Semen Ricini silkworm SAR element and pSK (+)-SAR clone's structure

Walk 1～1.2% agarose electrophoresis after DNA is purified, ³²P-α-dATP (Amersham product) mark rRNA gene fragment is made probe, and 5 ' EcoRI-SacII fragment of results of hybridization proof Semen Ricini silkworm rRNA gene is about 1, and 000bp can combine with nuclear skeleton, is defined as the SAR element of Semen Ricini silkworm.EcoRI-SacII site with this SAR inserts pBluescript (pSK+) is built into pSK (+)-SAR and clones (see figure 1), and the dna sequence dna composition of SAR element is seen Fig. 2.

Embodiment 2 Semen Ricini silkworm SAR element application are in the pLu-SAR Construction of eukaryotic

With SacII and EcoRI double digestion, the SAR fragment is downcut from pSK (+)-SAR plasmid, with the Klenow enzyme mend flat after, connect the BamHI joint, cut out sticky end with BamHI again after, insert the BamHI site of pLu plasmid, be built into pLu-SAR carrier for expression of eukaryon (see figure 3), the pLu plasmid is by U.S. Kohwi-Shigematsu, and the T. laboratory provides, be laboratory plasmid commonly used, the pLu plasmid contains penicillin resistance gene (Amp ^R) and neomycin resistance gene Neo ^RAs selection markers, contain the SV40 promotor and, determine that by the vigor of measuring the luciferin enzyme expression of exogenous gene just as the luciferin enzyme reporter gene (Luc) of foreign gene.The vigor of pLu plasmid expression Luc is very low, therefore can be used as contrast.With the BamHI site that Semen Ricini silkworm SAR element inserts the pLu plasmid, be positioned at 5 ' upstream of SV40 promotor, if the expression that this SAR element can enhancing gene, the vigor of luciferin enzyme just improves after testing.

Embodiment 3 Semen Ricini silkworm SAR elements strengthen the detection of foreign gene expression activity in eukaryotic cell

Carrier for expression of eukaryon pLu-SAR with embodiment 2 structures, and pLu in contrast, as each 1.5 μ g of the pAGLu plasmid of positive control, lipofection amine reagent (GIBCO company product) 6 μ l, rotaring copolymering NIH 3 T 3 cell, transfection method is undertaken by the operational manual that company provides, and the NIH3T3 cell count is about 2.5 * 10 ⁵/ 60mm plate screens with 0.7mg/mL G418 after 48 hours in transfection, obtain anti-G418 resistance clone after, the checking plasmid DNA be incorporated on the chromosomal DNA, detect the luciferin enzymic activity then.The authentication method that plasmid DNA is incorporated on the chromosomal DNA is: extract cell genomic dna with ordinary method, with PCR certification mark gene Neo ^RExistence, the PCR condition is: 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 1 minute 30 seconds, 30 circulations, 1% agarose electrophoresis is identified, the 790bpDNA amplified fragments occurs, illustrates that plasmid DNA is incorporated on the cell genomic dna.The activity of luciferin enzyme is measured with luciferin enzyme detection system (Promega company product), and method is seen Promega company product description, and LB9507 fluorescence calculating instrument is EG﹠amp; G Berthold company product.Measurement result as shown in Figure 4, the relative intensity of fluorescence reading is respectively: 3T3/pLu is 2033; 3T3/pAGLu is 14028; 3T3/pLu-SAR is 33954.3T3/pAGLu cell relative intensity of fluorescence is bigger seven times than 3T3/pLu cell, similar (Bode J to reported in literature, et al., Science, 1992,255:195～197), its relative intensity of fluorescence of 3T3/pLu-SAR cell has then strengthened 17 times, illustrates that SAR is applied to the ability that eukaryotic expression vector has very strong enhancing exogenous gene expression.

Claims (2)

1. an application of castor silkworm cell nucleus skeleton binding element, it is characterized in that this element is applied to constructing eukaryotic gene expression carrier, strengthens the expression of exogenous gene in eukaryotic cell. 2. the application of castor silkworm cell nuclear skeleton binding element as claimed in claim 1, is characterized in that described eukaryotic gene expression carrier is pLu-SAR, and SAR is positioned at the upstream of SV40 promotor, and wherein, SAR refers to nuclear skeleton binding district.

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