CN1319605A - Antitumour polypeptide and gene therapy vector composition - Google Patents

Abstract

The present invention relates to a polypeptide containing 53 amino acids (called p53 for short) and its gene recombinant retroviral vector. Said invention provides as antitumor p53 gene therapeutic composite mediated by viral vector, and said composite possesses the functions of regenerating blood vessel in target antitumor tissue and inhibiting growth and transfer of tumor.

Description

Tumor protein p53 and gene therapy vector composition thereof

The present invention relates to the Antioncogene therapeutic composition that a kind of new virus vector mediates, have the antitumor medium vessels new life that organizes of target and act on, thereby suppress growth of tumor and transfer.More particularly, relate to a kind of preparation and inside and outside application that contains the gene recombination retroviral vector and the modifier thereof of 53 amino acid polypeptides (being called for short p53).

New vessel in the noumenal tumour tissue is to keep the metabolic important leverage of tumour cell, is one of tumor growth and transfer conditio sune qua non.Because growth of tumor is that blood vessel is dependent, therefore,, and cut off the tumors of nutrients supply by the angiogenesis in the inhibition tumor tissues, just can reach antineoplastic purpose.At present, tumor treatment usually is to adopt secular combined chemotherapy and radiotherapy, and not only there is the drug resistance problem in whole body therapeutic, and can bring many serious or even lethal side effects to the patient.In recent years studies show that, the virus or the non-virus carrier of anti-angiogenic rebirth factor gene sequence will be carried, target importing tumor tissues or normal surrounding tissue, can pass through paracrine action mechanism, produce the angiogenesis supressor of high density at tumor by local, performance selectivity angiogenesis inhibiting and reach the purpose that suppresses tumor growth.Experimentation on animals proves, multiple angiogenesis there are inhibiting gene or gene fragment, as the responsive albumen of thrombus, vascular endothelial growth factor (VEGF) receptor domain mutant (flk-1), angiostatin (angiostatin), Endostatin (endostatin) and urokinase N-terminal fragment (ATF) etc. all have the effect of significant inhibition tumor growth.In addition,, also can effectively reduce the expression of the tumor by local FGF and the VEGF factor, the effect of performance effective antitumour at antisense technology (antisensetechnology) and/or the Ribozymes technology of fibroblast growth factor (FGF) and VEGF.In sum, the gene therapy of anti-angiogenic rebirth treatment tumour with its target, high local concentrations, avoid the resistance of tumour cell and farthest reduce the advantages such as toxic side effect of medicine, be expected to become in many antitumour treatments one of the most promising method.

The present invention's purpose is to seek a kind of gene target medicine of effective antitumour angiogenesis, and the toxic side effect that exists in the current clinical therapy of tumor is big to overcome, resistance is strong and shortcoming such as weak effect.Specifically, one of purpose is to provide a kind of p53 peptide factor that can suppress tumor growth and transfer, and with the constructed virus vector of p53 peptide gene as gene therapy compositions; Two of purpose is according to known p53 peptide nucleotide sequence, method by pcr amplification and sudden change, clone and change structure one and overlap p53 peptide based composition, set up the p53 peptide and change gene viruses, bacterium and the cell carrier system of structure polypeptide, screen and produce activated reorganization human P 53 peptide and virus vector (Fig. 3) thereof.

Content of the present invention and main points relate to a kind of 53 amino acid whose tumor protein p53s (be called for short p53 peptide) that contain, and with the constructed virus vector of the gene of p53 peptide.In order to inquire into effect and the mechanism thereof of p53 peptide in the treatment solid tumor, the present invention has made up the p53 recombinant retroviral vector, has set up expression-secretion type recombinant P 53 department of human head and neck tumour KB clone and corresponding nude mice solid tumor animal model.Result of study has confirmed that the p53 of retrovirus-mediated method has the neonate tumour blood vessel of inhibition and antineoplastic action.

The sequence of p53 peptide described in the invention is seen Fig. 1.In force, according to known array, designed primer and carried out pcr amplification, the primer sequence of amplification vector foreign gene: upstream primer: 5 '-CCCTTGAACCTCCTCGTTCGACC-3 ', downstream primer: 5 '-GAGCCTGGGGACTTTCCACACCC-3 '.With same primers as genomic dna is carried out pcr amplification.The amplification back obtains the series of genes fragment, and wherein p53 peptide gene sequence is seen Fig. 2.

P53 peptide cDNA is connected with signal peptide cDNA.The p53 cDNA sequence that signal peptide will be contained in mat EcoR I and BamH I site is reconstituted among the pLXSN/neo, obtains the pLXSNp52 recombinant retroviral vector.With pLXSNp53 is template, pcr amplification foreign gene p53, the PCR product separates through agarose gel electrophoresis, the low melting point glue purification reclaims respective strap, enzyme is cut rear clone in the plasmid vector M13mp19RF of identical double digestion, and transformed into escherichia coli JM109 is after the colourless plaque amplification cultivation of picking, extract double-stranded DNA and carry out enzyme spectrum analysis, extract single stranded DNA and carry out the dideoxy method determined dna sequence.Order-checking is pressed QIAGEN Plasmid Purification test kit description operation after identifying, purifying pLXSN-p52 is equipped with transfection package cell line PA317.Further press lipofecTAMINE TM test kit (GIBCOBRL) description operation, pLXSN-neo and pLXSNp53 are imported the PA317 cell respectively, G418 (GIBCOBRL) screening, collect G418 positive colony (PA317-neo and PA317-p53), educate altogether with PA317-neo and PA317-p52 nutrient solution supernatant and NIH3T3 respectively, virus titer is calculated in the G418 screening.Educate altogether with PA317-neo and PA317-p53 supernatant and KB cell respectively, G418 positive colony (KB-neo is collected in the G418 screening, KB-PF4, KB-p53), mixed culture PA317 G418 positive colony and KB G418 positive colony are identified and the inside and outside determination of activity respectively.

The present invention is through said process clone p53 peptide gene, make up recombinant retroviral vector, transfection PA317 package cell line obtains viral supernatant, with viral supernatant infected person incidence cancer KB cell, and integration has taken place in the goal gene and the KB cellular genome that import with PCR, RT-PCR and Western blotting methods analyst proof, obtained the KB cell strain of stably express recombinant P 53, and the transplanted tumor in nude mice level has been carried out multiparameter research in cell in vitro cultivation level and body.Experimental results show that: the importing of the p53 gene of (1) retroviral vector mediation does not influence the growth of KB tumour cell; (2) nutrient solution of the PA317-p53 cell of transfection and KB-p53 cell can suppress the propagation of endotheliocyte significantly; (3) the transplanted tumor in nude mice experimental observation is obviously slow to experimental group growth of xenografted speed, and the mice with tumor survival time obviously prolongs, and the density of new vessel obviously reduces in the transplanted tumor.The result confirms that the p53 peptide gene of retroviral vector mediation has the vascular endothelial cell of inhibition in-vitro multiplication, suppresses the effect of tumor growth in vivo then.

One of purpose of the present invention is to provide a kind of p53 peptide factor that can suppress tumor growth and transfer, and with the constructed viral carrying agent of p53 peptide gene as gene therapy compositions.

Two of purpose of the present invention is according to known p53 peptide nucleotide sequence, method by pcr amplification and sudden change, clone and change structure one and overlap p53 peptide based composition, set up the p53 peptide and change gene viruses, bacterium and the cell carrying agent system of structure polypeptide, screen and produce activated reorganization human P 53 peptide and gene viruses carrying agent thereof.

Embodiment 1, and virus titer is measured, the recombinant retroviral vector synergy is identified and Westernblotting analyzes

Respectively KB-neo, KB-p53 and wild type KB cell are grown in complete culture solution.When well-grown, prepare cell suspension, counting with fresh complete culture solution.Respectively with 2 * 10 ⁴/ ml is inoculated in 6 orifice plates and 96 orifice plates, and 3 porocytes of respectively getting every day in 6 orifice plates are counted, totally 6 days.The MTT method: cultivating 0,24,48,72 respectively, in the time of 96,120 hours, adding MTT (Sigma product), hatch 5 hours after, add DMSO (Sigma product), wavelength 570nm measures absorbancy.

Respectively KB-neo, KB-p53 and three kinds of KB cells of wild type KB are grown in complete culture solution.When well-grown, prepare cell suspension, counting with fresh complete culture solution.Be inoculated in 6 orifice plates by 200/ hole respectively, counting contains the colony number greater than 50 cells after about two weeks.PA317-neo and PA317-p53 nutrient solution supernatant are educated altogether with NIH3T3 respectively, and the virus titer of mensuration is 5 * 10 ⁶-8 * 10 ⁶CFU/ml.

Extract the genomic dna and the cell total rna of the KB cell of the packing cell PA317 of transfection and transfection respectively, identify the integration and the mRNA expression of foreign gene with PCR and RT-PCR method.The total RNA that extracts identifies through electrophoresis, 28S and 18S band clear (result slightly), with pLXSN multiple clone site both sides primer, PCR and RT-PCR amplification, the result is at PA317-neo and PA317-p53, and detecting purpose band: empty carrier-139bp in KB-neo and the KB-p53 cell, p53-389bp does not see special band in wild type KB cell and the wild type PA317 cell.Illustrate that recombinant retroviral vector successfully is integrated in the KB cellular genome through liposome-mediated.

Western blotting analytical results detects the albumen that molecular weight is 24kDa respectively in KB-p53 nutrient solution supernatant, the expression of secretor type p53 peptide is described.

Embodiment 2, the external influence to tumour cell and vascular endothelial cell growth of p53 peptide cDNA transfection

At first measure the growth curve of HUVE cell and wild type KB cell under the KB cell conditioned medium effect of cleer and peaceful transfection on the PA317 virus of transfection.Specifically, with HUVE cell and wild type KB cell (1 * 10 ⁴-2 * 10 ⁴/ m1) be inoculated in 96 orifice plates.Respectively PA317-neo and PA317-p53 cell are grown in nutrient solution simultaneously, when reaching 80% remittance sheet, growth uses the fresh nutrient solution that does not contain G418 instead, collect nutrient solution after 24 hours, 0.45 μ m membrane filtration cell debris, add in the nutrient solution of HUVE cell and wild type KB cell, continue to cultivate 2-3 cell growth cycle.The MTT method is measured and is found, the KB-p53 groups of cells compares the cell growth curve indifference with wild type KB cell and KB-neo cell respectively.PA317-p53 and PA317-neo compare HUVE cell proliferation restraining effect (Fig. 4).

Next measures the growth curve of HUVE cell and wild type KB cell under the KB cell culture fluid effect of transfection.Its method is inoculation HUVE cell and wild type KB cell (1 * 10 ⁴-2 * 10 ⁴/ m1) in 96 orifice plates.KB-neo and KB-p53 cell are grown in nutrient solution, when reaching 80% remittance sheet, growth uses the fresh complete culture solution that does not contain G418 instead, collected nutrient solution in 24 hours, 0.45 μ m membrane filtration cell debris, add in the nutrient solution of HUVE cell and wild type KB cell, continue to cultivate 2-3 cell growth cycle.The result confirms that KB-neo is consistent with the effect of PA317-neo and PA317-p53 virus supernatant to the influence of HUVE and the growth of wild type KB cell with KB-p53 nutrient solution supernatant.

Embodiment 3.The influence of p53 peptide cDNA transfection to growing in the implanted tumor cells body

Get female, 4-6 24 of the thymus gland immune deficiency nude mices (BALB/c-nu) (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute) in all ages, raise in the nude mice room and experiment.Be divided into four groups, 6 every group.Nude mice is carried out whole body exposure with the dosage of 4GY137Cs before the inoculating cell.In every subcutaneous KB-neo that inoculates respectively of nude mice right side forelimb, three kinds of KB cells (10 of KB-p53 and wild type KB ⁷Individual cell/every nude mice).Observed nude mice growth of tumor situation in per 3 days, and with the size of vernier caliper measurement, record tumour, { volume=(4/3) π (major diameter/2) (minor axis/2) (major diameter+minor axis)/4} is observed and is also write down death time of animal to calculate gross tumor volume by oval cubature formula.Repeat this experiment, and in the selected time, put to death animal, strip the knurl body, weigh, and the tumor tissues sample is done immunohistochemistry and analysis of Ultrastructure.Three kinds of KB cells of KB-wt, KB-neo and KB-p53 are inoculated in the average growth curve of the subcutaneous back of BALB/c-nu nude mice transplanted tumor respectively and see Fig. 5.The inoculation KB-neo Transplanted cells knurl speed of growth is the fastest, the inoculation KB-wt Transplanted cells knurl speed of growth and inoculation KB-neo Transplanted cells knurl speed of growth indifference (result slightly).The inoculation KB-p53 Transplanted cells knurl speed of growth is the slowest, compares with inoculation empty carrier cell, and significant difference (p=0.0001) is arranged.

Tumor cell inoculation 19 days, put to death animal, peel off the knurl piece, wild type KB knurl body weight in average is 1.6983 ± 0.345 grams, transfection empty carrier pLXSN-neo cDNA person knurl body weight in average is 1.943 ± 0.2534 grams, and the knurl body weight in average of transfection pLXSN-p53 cDNA group is 0.8017 ± 0.34 gram (the results are shown in Figure 6).

Fig. 6 represents that KB-wt, KB-neo and three kinds of KB cells of KB-p53 are inoculated in the survival curve of the subcutaneous back of BALB/c-nu nude mice animal respectively.The KB-p53 group is compared significant difference (p＜0.05) with the KB-neo group with the KB-wt group respectively.The KB-neo group is compared indifference with the KB-wt group.

Description of drawings:

Fig. 1: tumor protein p53 (p53 peptide) aminoacid sequence

Fig. 2: tumor protein p53 (p53 peptide) dna sequence dna

Fig. 3: the structure of recombinant P 53 peptide expression vector

Fig. 4: the recombinant virus supernatant is to the HUVE cel l proliferation

Fig. 5: the average growth curve of the subcutaneous KB Transplanted cells of nude mice knurl

Fig. 6: the recombinant P 53 peptide is to the growth-inhibiting effect of nude mice subcutaneous transplantation knurl

Claims (6)

1. a new tumor protein p53 material by the virus vector mediation, has target inhibition growth of tumor and transferance, it is characterized in that basic composition is 53 peptides or its active fragments of the listed aminoacid sequence of Fig. 1.

2. according to a kind of medicinal compositions of claim 1 manufacturing, it is characterized in that, contain right 1 described peptide molecule or its mutain.

3. the gene therapy of a tumor protein p53 is with gene recombined virus carrier and modifier thereof, it is characterized in that goal gene is made up of the listed dna sequence dna of Fig. 2 or its modifier.

4. according to claim 3, be used to express the plasmid of 53 peptides, it is characterized in that, contain right 2 described dna moleculars.

5. plasmid according to claim 4 is characterized in that described plasmid is selected from pET32c.

6. gene recombined virus carrier according to claim 3 is characterized in that, described virus can be retrovirus, also can be other virus.

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