CN102240405A - Apolipoprotein A-I (ApoA-I) milano genetic medicine - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to an apolipoprotein A-I (ApoA-I) milano genetic medicine. The ApoA-I milano genetic medicine is characterized by comprising an adenovirus vector for expressing a nucleotide sequence of an amino acid sequence shown as SEQ ID No.1. In the ApoA-I milano genetic medicine, a recombinant adenovirus vector containing an ApoA-I Milano gene is constructed by utilizing the characteristics of high infection efficiency, wide host range, high safety and the like of the adenovirus vector, so the ApoA-I milano genetic medicine is a new medicine for treating diabetes and atherosclerosis, and has a wide market prospect and a great economic benefit.

Description

ApoA-I milano genomic medicine

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of ApoA-I milano genomic medicine.

Background technology

Along with expanding economy, the diabetes incidence rate rises year by year, and coronary heart disease is the underlying cause of death of diabetes, and almost 90% diabetes patient dies from atherosclerosis (especially arteria coronaria) complication.Known HDL(high density lipoprotein) is a kind of inverse indicators of following cardiovascular event, improves blood plasma HDL level and can alleviate atherosclerosis and reverse pathological changes.

ApoA-I is the main apolipoprotein of HDL, and some functions of HDL mainly have ApoA-I to carry out.In the mankind, insulin resistant is closely related with the reduction of blood middle-high density lipoprotein (HDL) and main component ApoA (ApoA-I) content thereof.Studies show that recently hyperglycemia and Hyperinsulinism have appearred in the ApoA-I knock out mice; glucose tolerance experiment also shows the infringement that the glucose metabolism of ApoA-I knock out mice is subjected to; the early stage disease that type occurred; ApoA-I is by combine activation AMPK signal path with part in the born of the same parents of adiponectin receptor; improve intravital blood glucose balance, thereby in type, brought into play protective effect.ApoA-I is directly related with the insulin sensitivity of carbohydrate metabolism and body in this result of study prompting, and the concentration that improves apoA-I in the body may be significant for the pathophysiological process of diabetes-alleviating.

ApoA-I milano is the genetic mutant of wild type ApoA-I, and wherein No. 173 serine replaced by arginine, and forms homodimer and heterozygote dimer ApoA-II.The HDL metabolism that carries ApoA-I milano is faster, and discovers that ApoA-I milano has the inhibition atherosclerosis of aorta, reduces effects such as intimal thickening, but combines anticoagulant with phosphide, promotes fibrinolytic, postpones artery thrombosis etc.So may becoming, ApoA-I milano is fit to very much treatment diabetes and atherosis medicine thereof.

Summary of the invention

Technical problem to be solved by this invention provides a kind of ApoA-I milano genomic medicine, provides new medicine for treating diabetes and atherosis medicine thereof.

For this reason, the invention discloses a kind of ApoA-I milano genomic medicine, it is characterized in that it comprises the adenovirus vector of expression nucleotide sequence of aminoacid sequence shown in SEQ ID NO.1.

In one embodiment, described nucleotide sequence is shown in SEQ ID NO.2.

In one embodiment, described adenovirus vector is pShuttle CMV.

The present invention utilizes adenovirus vector efficiency of infection height, host range extensively reaches characteristics such as safety is better, structure contains ApoA-I milano gene recombinant adenovirus vector, can be treatment diabetes and atherosis medicine thereof new medicine is provided, have vast market prospect and bigger economic benefit.

Description of drawings

Fig. 1 is that EcoR I enzyme action is identified pShuttle CMV-A I milano electrophoretogram;

Fig. 2 is that Hind III enzyme action is identified pAd CMV-A I milano electrophoretogram;

Fig. 3 is that Not I and EcoR I enzyme action are identified pShuttle CMV-GFP electrophoretogram;

Fig. 4 is that Hind III enzyme action is identified pAd CMV-GFP electrophoretogram;

Fig. 5 ApoA-I milano Western blotting figure that behaves.

The specific embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.

Embodiment1.ApoA-I milano plasmid construction

The total RNA reverse transcription of human blood adopts RT-PCR amplification human apolipoprotein A I(ApoA I) cDNA, order-checking confirms that the back is mutated into ApoA I milano-cDNA with Stratagene QuikChange rite-directed mutagenesis test kit with ApoA I-cDNA.

The mutant primer sequence as follows

Upstream: 5 '-CTGGGCGAGGAGATGTGCGACCGCGCGCGC-3 '

Downstream: 5 '-GCGCGCGCGGTCGCACATCTCCTCGCCCAG-3 '

Sudden change back cDNA double digestion is connected into pDNR-LIB-PA, transformed into escherichia coli, choose clone, checking order is accredited as pDNR-LIB-ApoA-I milano-PA plasmid and is used for follow-up test.

Embodiment 2. contains the preparation of genes of interest pShuttle plasmid

2.1 contain the preparation of the pShuttle CMV-A I milano plasmid of ApoA-I milano expression cassette

PDNR-LIB-apoA I milano-PA plasmid through 1% agarose gel electrophoresis, reclaims test kit with gel and collects the segment that purification contains ApoA I milano gene behind restricted enzyme Sal I and Hind III double digestion.PShuttle CMV carries out double digestion with restricted enzyme Sal I and Hind III, reclaims the plasmid vector segment, carries out coupled reaction with ApoA-I milano segment.Connect product and be converted in the DH5 α competence escherichia coli, transformed bacteria screens with kanamycin, and carries out EcoR I enzyme action and identify.The correct strain of enzyme action evaluation is preserved, and prepared pShuttle CMV-A I milano plasmid in a large number.

2.2 contain the preparation of egfp expression frame pShuttle CMV-GFP plasmid

The pAAV-EFla-GFP-PA plasmid is after restricted enzyme Kpn I and Xba I are carried out double digestion, and purification reclaims and contains green fluorescent protein (green fluorescent protein, GFP) gene purpose segment.After pShuttle CMV carried out double digestion with restricted enzyme Kpn I and Xba I, purification reclaimed the plasmid vector fragment, carries out coupled reaction with the GFP fragment.To connect product and be transformed in the competence DH5 α escherichia coli, transformed bacteria is cultivated the extraction plasmid DNA in a small amount and is carried out the enzyme action evaluation with restricted enzyme Hind III with kanamycin screening monoclonal bacterium colony.The correct strain of enzyme action evaluation is preserved, and prepared pShuttle CMV-GFP plasmid in a large number.

Embodiment 3. contains the preparation of genes of interest pAd plasmid

PShuttle plasmid (pShuttle CMV-A I milano and pShuttle CMV-GFP) obtains corresponding pAd plasmid (pAd CMV-A I milano and pAd CMV-GFP) through recombinating in competence BJ5183 escherichia coli with the pAdeasy-1 plasmid behind the Pme I linearization for enzyme restriction.Enzyme action is identified that correct plasmid changes DH5 α antibacterial over to and prepares in a large number and store.

Embodiment 4. contains the preparation of genes of interest adenovirus seed culture of viruses

The pAd plasmid (pAd CMV-A I milano and pAd CMV-GFP) that contains genes of interest is behind Pac I linearization for enzyme restriction, be transfected in the HEK293 cell of low generation, cultivate after 8-12 days, collecting cell and supernatant thereof after the tangible cytopathic effect occur, obtain the adenovirus seed culture of viruses (AdV-A I milano and AdV-GFP) that correspondence contains genes of interest.

Embodiment 5. contains the evaluation of the adenovirus vector of ApoA I milano gene

Obtain to contain ApoA I milano encoding gene from pDNR-LIB-apoA I milano-PA plasmid, insert pShuttle CMV shuttle plasmid, obtain pShuttle CMV-A I milano.This plasmid and the reorganization of pAdeasy-1 plasmid obtain pAd CMV-A I milano.(1 is EcoR I enzyme action pShuttleCMV-A I milano among Fig. 1 to utilize plasmid sequence mapping, the pShuttle CMV-A I milano of structure to obtain 4921bp and two purpose segments of 3486bp section behind EcoR I enzyme action; 2 is DNA marker), (1 is Hind III enzyme action pAd CMV-A I milano among Fig. 2 to obtain 8010bp, 5913bp, 5322bp, 4597bp, 3010bp, 2985bp, 2945bp, 2081bp and 75bp purpose fragment behind the pAd CMV-A I milano enzyme action; 2 is DNA marker).

Embodiment 6. contains the evaluation of the adenovirus vector of green fluorescence protein gene

Obtain to contain the GFP encoding gene from the pAAV-EFla-GFP-PA plasmid, insert pShuttle CMV shuttle plasmid, obtain pShuttle CMV-GFP, this plasmid and the reorganization of pAdeasy-1 plasmid obtain pAd CMV-GFP.Utilize the plasmid sequence mapping, obtain 1864bp and two purpose fragments of 747bp behind the pShuttle CMV-GFP enzyme action, (1 is Not I and EcoR I double digestion pShuttle CMV-GFP among Fig. 3 to obtain 8010bp, 5322bp, 4932bp, 4597bp, 3010bp, 2980bp, 2945bp, 2081bp and 1964bp purpose fragment behind the pAd CMV-GFP enzyme action; 2 is 1 to identify pAd CMV-GFP for Hind III enzyme action among DNA marker, Fig. 4; 2 is DNA marker).

The evaluation of embodiment 7. recombinant viruses

The recombinant virus plasmid pAd CMV-A I milano and the pAd CMV-GFP that successfully construct use the hydrolysis of Pac I enzyme action respectively, obtain the linearisation double-strandednucleic acid, and transfection HEK293 cell can be packed becomes adenovirus AdV-A I milano and AdV-GFP.CPE can appear in the HEK293 cell of transfection success, proves that virus is in the success of cell inner packing.After amplification culture, collect the recombinant adenovirus of the centrifugal acquisition purification of employing virus cracking liquid.After in the adenovirus AdV-A I virus injection mice body behind the purification 1 day, (1 is unloaded virus particle among Fig. 5 to detect human apolipoprotein A I milano by Western blotting monitoring in serum; 2 is ApoA-I milano adenovirus).

Scope of the present invention is not subjected to the restriction of described specific embodiments, and described embodiment also comprises the method and the component of functional equivalent only as the single example of illustrating various aspects of the present invention in the scope of the invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to above description and accompanying drawing.Described improvement also falls within the scope of appended claims.Every piece of list of references mentioned above is listed this paper in as a reference all in full.

SEQUENCE?LISTING

 

＜110〉Shanghai Tenth People's Hospital

 

＜120〉description sequence table

 

＜130〉a kind of ApoA-I milano genomic medicine

 

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Met?Lys?Ala?Ala?Val?Leu?Thr?Leu?Ala?Val?Leu?Phe?Leu?Thr?Gly?Ser

1 5 10 15

 

 

Gln?Ala?Arg?His?Phe?Trp?Gln?Gln?Asp?Glu?Pro?Pro?Gln?Ser?Pro?Trp

20 25 30

 

 

Asp?Arg?Val?Lys?Asp?Leu?Ala?Thr?Val?Tyr?Val?Asp?Val?Leu?Lys?Asp

35 40 45

 

 

Ser?Gly?Arg?Asp?Tyr?Val?Ser?Gln?Phe?Glu?Gly?Ser?Ala?Leu?Gly?Lys

50 55 60

 

 

Gln?Leu?Asn?Leu?Lys?Leu?Leu?Asp?Asn?Trp?Asp?Ser?Val?Thr?Ser?Thr

65 70 75 80

 

 

Phe?Ser?Lys?Leu?Arg?Glu?Gln?Leu?Gly?Pro?Val?Thr?Gln?Glu?Phe?Trp

85 90 95

 

 

Asp?Asn?Leu?Glu?Lys?Glu?Thr?Glu?Gly?Leu?Arg?Gln?Glu?Met?Ser?Lys

100 105 110

 

 

Asp?Leu?Glu?Glu?Val?Lys?Ala?Lys?Val?Gln?Pro?Tyr?Leu?Asp?Asp?Phe

115 120 125

 

 

Gln?Lys?Lys?Trp?Gln?Glu?Glu?Met?Glu?Leu?Tyr?Arg?Gln?Lys?Val?Glu

130 135 140

 

 

Pro?Leu?Arg?Ala?Glu?Leu?Gln?Glu?Gly?Ala?Arg?Gln?Lys?Leu?His?Glu

145 150 155 160

 

 

Leu?Gln?Glu?Lys?Leu?Ser?Pro?Leu?Gly?Glu?Glu?Met?Arg?Asp?Arg?Ala

165 170 175

 

 

Arg?Ala?His?Val?Asp?Ala?Leu?Arg?Thr?His?Leu?Ala?Pro?Tyr?Ser?Asp

180 185 190

 

 

Glu?Leu?Arg?Gln?Arg?Leu?Ala?Ala?Arg?Leu?Glu?Ala?Leu?Lys?Glu?Asn

195 200 205

 

 

Gly?Gly?Ala?Arg?Leu?Ala?Glu?Tyr?His?Ala?Lys?Ala?Thr?Glu?His?Leu

210 215 220

 

 

Ser?Thr?Leu?Ser?Glu?Lys?Ala?Lys?Pro?Ala?Leu?Glu?Asp?Leu?Arg?Gln

225 230 235 240

 

 

Gly?Leu?Leu?Pro?Val?Leu?Glu?Ser?Phe?Lys?Val?Ser?Phe?Leu?Ser?Ala

245 250 255

 

 

Leu?Glu?Glu?Tyr?Thr?Lys?Lys?Leu?Asn?Thr?Gln

260 265

 

 

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cccagagccc?ctgggatcga?gtgaaggacc?tggccactgt?gtacgtggat?gtgctcaaag 180

 

acagcggcag?agactatgtg?tcccagtttg?aaggctccgc?cttgggaaaa?cagctaaacc 240

 

taaagctcct?tgacaactgg?gacagcgtga?cctccacctt?cagcaagctg?cgcgaacagc 300

 

tcggccctgt?gacccaggag?ttctgggata?acctggaaaa?ggagacagag?ggcctgaggc 360

 

aggagatgag?caaggatctg?gaggaggtga?aggccaaggt?gcagccctac?ctggacgact 420

 

tccagaagaa?gtggcaggag?gagatggagc?tctaccgcca?gaaggtggag?ccgctgcgcg 480

 

cagagctcca?agagggcgcg?cgccagaagc?tgcacgagct?gcaagagaag?ctgagcccac 540

 

tgggcgagga?gatgcgcgac?cgcgcgcgcg?cccatgtgga?cgcgctgcgc?acgcatctgg 600

 

ccccctacag?cgacgagctg?cgccagcgct?tggccgcgcg?ccttgaggct?ctcaaggaga 660

 

acggcggcgc?cagactggcc?gagtaccacg?ccaaggccac?cgagcatctg?agcacgctca 720

 

gcgagaaggc?caagcccgcg?ctcgaggacc?tccgccaagg?cctgctgccc?gtgctggaga 780

 

gcttcaaggt?cagcttcctg?agcgctctcg?aggagtacac?taagaagctc?aacacccagt 840

 

gaggcgcccg?ccgccgcccc?ccttcccggt?gctcagaata?aacgtttcca?aagtggg 897

 

 

Claims (3)

1. ApoA-Imilano gene medicine is characterized in that it comprises the adenovirus vector expressing the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.1. 2. ApoA-Imilano gene medicine according to claim 1, characterized in that said nucleotide sequence is as shown in SEQ ID NO.2. 3. ApoA-Imilano gene medicine according to claim 1, characterized in that said adenoviral vector is pAd CMV.

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