CN103333891A - Small interference RNA aiming at HPV16E7 gene, and application thereof - Google Patents

Abstract

The invention relates to a double-stranded RNA (dsRNA) for preventing and treating human papilloma virus 16 subtype (HPV16) infections. The dsRNA comprises a fragment formed by 19-25 nucleotides in length and is largely complementary to at least one part selected from HPV16E7 gene. The invention also relates to a medicinal composition containing the dsRNA and a pharmaceutically acceptable vector, a method for applying the dsRNA and the medicinal composition in preventing and treating the HPV16 infections and diseases caused by HPV16 infections, and a method for applying the dsRNA and the medicinal composition in inhibiting the expression of the HPV16 E7 gene in a cell.

Description

SiRNA and application thereof at the HPV16E7 gene

The application be that September 27, application number in 2011 are 201110288582.4 the applying date, denomination of invention divides an application for the application for a patent for invention of " at siRNA and the application thereof of HPV16E7 gene ".

Technical field

The present invention relates to double stranded RNA (dsRNA), and relate to it and disturb (RNAi) to be used for the purposes of prevention and the caused relative disease for the treatment of human papilloma virus 16 hypotype (HPV16) infection by RNA, wherein said disease is cancers such as cervical cancer, carcinoma vulvae, the esophageal carcinoma, mammary cancer, lung cancer for example, and by diseases such as the caused precancerous lesion of HPV16 and Genital wartss.

Background technology

1998, Andrew Z.Fire and Craig C.Mello found the mechanism of action that RNA disturbs in the body jointly, and had obtained Nobel Prize in Physiology or Medicine jointly in 2006.Thereby a fan gate has been opened in the research and development that are the medicine of new generation (RNA disturbs the class medicine) of serious diseases such as opposing virus, cancer.This RNA disturbs that class medicine has mode of action novelty, mechanism of action is clear and definite, targeting is strong and advantage such as side effect is little.

Human papillomavirus's (Human Papillomavirus is called for short HPV) belongs to the papovaviridae Papillomavirus, is a kind of small-sized nonencapsulated virion, and is spherical in shape, the icosahedron symmetry, and the about 45-55nm of diameter, the core of virus is double-stranded DNA; Viral capsid is made up of 72 shell particulates that two kinds of structural protein constitute.Viral DNA comprises about 8000 base pairs, comprising 8 early stage open reading frames (E1-E8), 2 single open reading frames in late period (L1 and L2) and 1 long control district of non-coding (LCR).In the open reading frame, E6 and the growth-stimulating of E7 gene pairs cell are the most important in early days, and the E7 albumen of E7 coding just can cause the cervical epithelial cells immortalization separately.And late period reading frame L1 and encode the respectively main and less important capsid protein of HPV of L2 gene, be assembled into the capsid of HPV.

HPV is one of most popular sexually transmitted disease source in the world, also is the important human tumor virus of a class simultaneously.Identified at present the hypotype that surpasses 100 kinds of HPV viruses.Between HPV is various common antigen is arranged, be present in L1 albumen conservative region, it is special that these antigens have genus.Position, epithelium place according to its infection: be divided into skin epitheliated type HPV and mucous epithelium type HPV.

Definite and most important HPV related neoplasms is cervical cancer the earliest, and its sickness rate is only second to mammary cancer, is the killer of serious threat women's health.New cases about 490,000 examples are arranged in the annual world wide according to statistics approximately, wherein about 130,000 occur in China, have every year 270,000 people to die from this disease approximately, about 50,000th, Chinese.Subsequently, find that more the generation of malignant tumours such as carcinoma vulvae, the esophageal carcinoma, mammary cancer even lung cancer, development all infect relevant with HPV.So far in more than the 100 kind of HPV hypotype of Fa Xianing, high carcinogenic HPV hypotype (being high-risk HPV) has kind more than 10, comprises HPV16,18,31,58 etc.Think that at present the HPV infection is necessary but non-sufficient to cervical cancer.Show that according to the study 99.7% cervical cancer patient exists HPV to infect.

The women's initial stage symptom that has infected HPV is not obvious.By based on to the Pap of abnormal cervical cells Histological assessment test and existence and the definite HPV hypotype that can determine HPV based on PCR and the hybridization technique of nucleic acid.Abnormal cervical cells is classified as low SIL (LSIL) and HSIL (HSIL).Wherein LSIL comprises that cervical intraepithelial neoplasia becomes the cell of one-level (CIN I); HSIL comprises that cervical intraepithelial neoplasia becomes the cell of secondary (CIN II) and three grades (CIN III).

The medicine and the therapy that are used for the treatment of the HPV infection at present mainly contain: 1, resin of podophyllium (podophyllin); 2, podophyllotoxin podophyllotoxin (podofilox); 3, trichoroacetic acid(TCA) (TCA)/dichloro acetic acid (BCA); 4, psychrotherapy; 5, excision; 6, Interferon, rabbit (IFN); 7, Imiquimod hniquimod[1-(2-methylpropyl)-1H-imidazo (4,5-quinolin-4-amine)]; 8,5 FU 5 fluorouracil/suprarenin; 9, acyclic nucleotide acid-like substance Cidofovir (HPMPC:[5]-1-[3-hydroxy-2-phosphonylmethoxy-propyl]-cytosine) etc.Yet these of Ying Yonging not only specificity antiviral and other antitumor class medicine is not strong clinically, and side effect is big.There are not at present a kind of special, generation and development that medicine that suppress HPV efficiently reduces even suppress its associated cancer in the world.

Summary of the invention

For addressing the above problem, the invention provides the HPV E7 genetic expression that causes the optimum and neoplasm of cell by the small RNA molecules in inhibiting, prevent and treat the solution that HPV16 infects relative disease.

In a first aspect of the present invention, the application of small RNA molecule in the preparation of expressing in cell for the preparation of inhibition HPV16E7 gene is provided, described small RNA molecule instructs the shearing of the HPV16E7mRNA sequence that exists in the described cell, thereby realizes described inhibition.In the present invention, described small RNA molecule comprises just nucleic acid fragment and antisense nucleic acid fragment, described just nucleic acid fragment and described antisense nucleic acid fragment contain complementary region, and complementary region can form the double-strandednucleic acid structure, and this double-strandednucleic acid can suppress the expression of HPV16E7 gene in cell.The just nucleic acid fragment of small RNA may reside on two different nucleic acid chains with the antisense nucleic acid fragment, also may reside on same the nucleic acid chains.When just nucleic acid fragment and antisense nucleic acid fragment laid respectively on two chains, at least one chain of small RNA had the 3 ' overhang that length is 0 to 6 Nucleotide, and two chains all have the 3 ' overhang that length is 2-3 Nucleotide under the preferable case.When just nucleic acid fragment and antisense nucleic acid fragment were present on same the nucleic acid chains, this small RNA was the hair clip type single stranded nucleic acid molecule under the preferable case, and wherein the complementary region of just nucleic acid fragment and antisense nucleic acid fragment forms the double-strandednucleic acid structure.In the above-mentioned small RNA molecule, the length of just nucleic acid fragment and antisense nucleic acid fragment is 19 to 29 Nucleotide, can be 19,20,21,23,25,27 and 29, is preferably 19,21,25,27 Nucleotide.Above-mentioned small RNA molecule can directly be imported in the described cell, also can be that the nucleotide sequence with this small RNA molecule of coding produces in cell after importing described cell; Described cell is preferably mammalian cell, more preferably the human cell.Above-mentioned cell can exsomatize, and as clone, also may reside in the mammalian body, in human body.This human body is the patient who suffers from the HPV infection symptoms, and described small RNA molecule is applied enough amounts to realize the treatment to described symptom, and described HPV infection symptoms is cervical cancer or pointed condyloma.

Second aspect present invention provides a kind of HPV16E7 gene small molecule interference nucleic acid target sequence of separation, wherein said target sequence is that the mRNA (cDNA) of HPV16E7 goes up arbitrarily 19-30 nucleotide sequence continuously, preferably, described target sequence is any sequence among the SEQID NO:201-270 in the table 1.

In a third aspect of the present invention, a kind of small RNA molecule is provided, it instructs the shearing of the HPV16E7mRNA sequence that exists in the cell, thereby realize inhibition that HPV16E7 is expressed, under the preferable case this small RNA molecule comprise with table 1 in the sequence of any sequence complementation among the SEQ ID NO:201-270.Nucleotide all in the above-mentioned small RNA molecule can be the natural Nucleotide without chemically modified, and also can have a Nucleotide at least is the Nucleotide of chemically modified, and the chemically modified mode is selected from least a in the following modification:

(1) to connecting the modification of the phosphodiester bond of Nucleotide in the nucleotide sequence of described small RNA molecule;

(2) to the modification of 2 '-OH of the ribose in the nucleotide sequence of described small RNA molecule;

(3) to the modification of the base in the nucleotide sequence of described small RNA molecule.

Can effectively reduce the expression amount of HPV16E7mRNA after the cells contacting of small RNA of the present invention and expression HPV16E7mRNA

The present invention also provides a kind of composition that comprises above-mentioned each described small RNA molecule and pharmaceutically acceptable carrier at last, and under the preferable case, pharmaceutically acceptable carrier is liposome or high molecular polymer.

Beneficial effect of the present invention

Small RNA molecule at HPV16E7 genetic expression provided by the invention can suppress the HPV16E7 expression of gene efficiently, specifically, has lower toxic side effect simultaneously, can be used for preparing the medicine that suppresses HPV16E7 genetic expression.

Embodiment

The present invention can cause the HPV16E7 genetic expression of the optimum and neoplasm of cell by RNA conflicting mode silence, prevents and treats HPV16 and infect this problem of relative disease.The HPV16E7 gene is also referred to as the HPV target gene sometimes among the present invention.

Small RNA molecule provided by the invention comprises having the nucleic acid fragment (antisense fragment) that length is lower than 30 Nucleotide and is complementary to the zone of at least part of HPV said target mrna transcript substantially.The use of these small RNA molecules can disturb the process of (RNAi) to cause HPV target gene mRNA to be sheared by being called as RNA.Small RNA molecule provided by the invention can be natural not modified nucleic acid molecule, also can carry out chemically modified at least one Nucleotide wherein.According to the present invention, described chemically modified is at least a in the following modification:

(1) to connecting the modification of the phosphodiester bond of Nucleotide in the nucleotide sequence of described siRNA;

(2) to the modification of 2 '-OH of ribose in the nucleotide sequence of described siRNA;

(3) to the modification of base in the nucleotide sequence of described siRNA.

Described chemically modified is conventionally known to one of skill in the art, and the modification of described phosphodiester bond refers to the oxygen in the phosphodiester bond is modified, and comprises the thiophosphoric acid modification, as shown in Equation 1; Modify with borine phosphoric acid salt, as shown in Equation 2.Two kinds of modifications can both be stablized the siRNA structure, keep high specific and the high-affinity of base pairing.

Described ribose is modified the modification that refers to 2 '-OH in the Nucleotide pentose,, introduces some substituting group in the hydroxy position of ribose that is, and for example, 2 '-fluoro is modified, as shown in Equation 3; 2 '-oxygen methyl is modified, shown in 4; 2 '-oxygen ethylidene methoxyl group is modified, as shown in Equation 5; 2,4 '-dinitrophenol(DNP) is modified, as shown in Equation 6; Lock nucleic acid (LNA), as shown in Equation 7; 2 '-amido modified, as shown in Equation 8; 2 '-deoxidation is modified, as shown in Equation 9.

Described base modification refers to the base of Nucleotide is modified, for example, 5 '-the bromouracil modification, as shown in Equation 10; 5 '-the iodouracil modification, as shown in Equation 11; The N-methyl uracil is modified, as shown in Equation 12; 2,6-diaminopurine is modified, as shown in Equation 13.

These modifications can increase the bioavailability of described small RNA molecule, and the affinity of raising and target sequence strengthens the ability of resisting the nuclease hydrolysis in cell.

In addition, in order to promote the small RNA molecule to enter cell, can be on the basis of above modification, introduce lipophilic group such as cholesterol at the end of the positive-sense strand of small RNA molecule and be beneficial to have an effect by the cytolemma and the intracellular mRNA that are constituted by lipid bilayer.

By the test based on cell and animal level, the contriver confirms these small RNA molecules mediate rna i effectively, causes the remarkable inhibition of HPV16E7 genetic expression, and suppresses to implant the growth of knurl body.Therefore, comprise small RNA molecule of the present invention and can treat the pathologic process that is infected mediation by HPV by target HPV target gene.

The preparation method of small RNA molecule provided by the invention comprises that sequences Design and sequence are synthetic.

The synthetic method that can adopt chemosynthesis of described small RNA molecular sequences, it is synthetic that perhaps the synthetic biotech company of nucleic acid is specialized in trust, synthesizes as entrusting Shanghai Ai Bosi bio tech ltd.

In general, the method for described chemosynthesis comprises following four processes: (1) oligomerization ribonucleotide synthetic; (2) deprotection; (3) purifies and separates; (4) desalination.

For example, it is as follows to have concrete steps of siRNA chemosynthesis of nucleotide sequence shown in the HPV16E7si01:

(1) the oligomerization ribonucleotide is synthetic

In that automated DNA/the RNA synthesizer (for example, Applied Biosystems EXPEDITE8909) goes up the RNA that sets synthetic 1 mmole, the coupling time of setting each circulation simultaneously is 10-15 minute, initiator is the dimethoxy of 5 '-O--thymidine upholder that solid phase connects, first circulates in and connects a base on the solid support, then in the n time (19 〉=n 〉=2) circulation, the base that connects the n-1 time circulation connects a base, repeats this circulation until finishing the synthetic of whole nucleotide sequences.

(2) deprotection

The solid support that is connected with siRNA is joined in the test tube, and in this test tube, add ethanol/ethamine (volume ratio is 1: 3) of 1 milliliter, sealing then, place 55-70 ℃ of incubator, hatched 2-30 hour, taking-up is connected with the solid support of siRNA and with distilled water drip washing 2 times (each 1 milliliter), collects elutriant, and at room temperature dry 30 minutes.Then, add the tetrahydrofuran solution (1M) of 1 milliliter of tetrabutyl ammonium fluoride, room temperature was placed 4-12 hour, added 2 milliliters of ethanol again, and collecting precipitation namely obtains the crude product of siRNA.

(3) purifies and separates

It is in 1 mole/milliliter the ammonium acetate solution that the crude product of the siRNA that obtains is dissolved in 2 ml concns, separates by the C18 high pressure liquid chromatography then, obtains the siRNA product of purifying.

(4) desalination

Be siRNA product 2-4 time (each 2 milliliters) of the aqueous ethanolic solution washing purifying of 75 weight % with concentration, removing salt, and drying under the room temperature.Then with oligomerization Yeast Nucleic Acid mixed dissolution (10mM Tris in the damping fluid of 1-2 milliliter of positive-sense strand and antisense strand, pH=7.5-8.0,50mM NaCl), this solution is heated to 95 ℃, slowly this solution is cooled to room temperature then, and kept room temperature 16-22 hour, obtain containing the solution of siRNA.

Originally discover, behind above-mentioned siRNA transfectional cell, can effectively suppress the expression of the interior HPV16E7RNA of cell and animal heteroplastic transplantation knurl and albumen.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment one: design HPV E7 small RNA sequence

HPV E7mRNA sequence obtains from the biomedical website ncbi database (http://www.ncbi.nlm.nih.gov/) of the U.S.'s state-run commune hospital construction, in siDESIGN Center and the online design specificity of the DSIR siRNA sequence at this gene.The principle of choosing the small RNA molecular sequences by online software definition is: mainly based on the linear model that is combined with small RNA molecular sequences and target sequence feature.The terminal high stability of small RNA molecule 5 ' may be blocked small RNA molecule positive-sense strand and be loaded into RNA and induce silencing complex (RISC); Small RNA molecule antisense strand 5 ' terminal low stability may promote small RNA molecule antisense strand to be loaded into RISC; And the low stability of small RNA molecule region intermediate can promote RISC-small RNA molecular complex to discharge.

Principle of design according to above-mentioned small RNA molecular sequences, design 70 Smal linterference ribonucleic acid sequences at HPV16E7 (siRNA) (seeing the following form 2) of the dsRNA HPV16E7si01～HPV16E7si70 that comprises positive-sense strand and antisense strand, described small RNA molecule comprises 2 deoxythymidylic acids (T) at 3 ' end of positive-sense strand and antisense strand.Among the described dsRNA, HPV16E7si01～HPV16E7si60 total length is the structure that has two Nucleotide 3 ' protruding terminuses of 21bp, HPV16E7si61～HPV16E7si70 total length is the structure that has two Nucleotide 3 ' protruding terminuses of 27bp, and HPV16E7si71～HPV16E7si80 total length is the flat end structure of 25bp.Last 10 dsRNA are the structure that HPV16E7si01 in the his-and-hers watches 2～HPV16E7si10 modifies in the table 2, overstriking is represented in the positive-sense strand is Nucleotide through modifying, ome represents the modification of 2 '-methoxyl group, and F represents 2 '-deoxidation-2 '-fluoro modification, and S represents the phosphorothioate bond modification.

Table 1.HPV16E7 gene target sequence

The sequence title	Target sequence (5 '-3 ')	Corresponding sequence number
			HPV16E7?01	GATTTGCAACCAGAGACAA	SEQ?ID?N0：201
HPV16E7?02	GGACAGAGCCCATTACAAT	SEQ?ID?NO：202
			HPV16E7?03	GCACACACGTAGACATTCG	SEQ?ID?NO：203
HPV16E7?04	GCTCAGAGGAGGAGGATGA	SEQ?ID?NO：204
			HPV16E7?05	CAGAGGAGGAGGATGAAAT	SEQ?ID?NO：205
HPV16E7?06	AGAGGAGGAGGATGAAATA	SEQ?ID?NO：206
			HPV16E7?07	AGGAGGAGGATGAAATAGA	SEQ?ID?NO：207
HPV16E7?08	GGATGAAATAGATGGTCCA	SEQ?ID?NO：208
			HPV16E7?09	CGTACAAAGCACACACGTA	SEQ?ID?NO：209
HPV16E7?10	GCAATTAAATGACAGCTCA	SEQ?ID?NO：210
			HPV16E7?11	AGACATTCGTACTTTGGAA	SEQ?ID?NO：211
HPV16E7?12	GCATGGAGATACACCTACA	SEQ?ID?NO：212
			HPV16E7?13	CGGACAGAGCCCATTACAA	SEQ?ID?NO：213
HPV16E7?14	AAGCACACACGTAGACATT	SEQ?ID?NO：214
			HPV16E7?15	TCTCTACTGTTATGAGCAA	SEQ?ID?NO：215
HPV16E7?16	TGAAATAGATGGTCCAGCT	SEQ?ID?NO：216
			HPV16E7?17	AGATTTGCAACCAGAGACA	SEQ?ID?NO：217
HPV16E7?18	GGAGGAGGATGAAATAGAT	SEQ?ID?NO：218
			HPV16E7?19	ACACCTACATTGCATGAAT	SEQ?ID?NO：219
HPV16E7?20	GGACAAGCAGAACCGGACA	SEQ?ID?NO：220
			HPV16E7?21	GACAGAGCCCATTACAATA	SEQ?ID?NO：221
HPV16E7?22	CACCTACATTGCATGAATA	SEQ?ID?NO：222
			HPV16E7?23	TGTTAGATTTGCAACCAGA	SEQ?ID?NO：223
HPV16E7?24	AGATACACCTACATTGCAT	SEQ?ID?NO：224
			HPV16E7?25	GTACAAAGCACACACGTAG	SEQ?ID?NO：225
HPV16E7?26	CTTTGGAAGACCTGTTAAT	SEQ?ID?NO：226
			HPV16E7?27	TCTACTGTTATGAGCAATT	SEQ?ID?NO：227
HPV16E7?28	AAGCAGAACCGGACAGAGC	SEQ?ID?NO：228
			HPV16E7?29	GTTAATGGGCACACTAGGA	SEQ?ID?NO：229

[0053]?

HPV16E7?30	GGAGGATGAAATAGATGGT	SEQ?ID?NO：230
			HPV16E7?31	AATGGGCACACTAGGAATT	SEQ?ID?NO：231
HPV16E7?32	ATGACAGCTCAGAGGAGGA	SEQ?ID?NO：232
			HPV16E7?33	TCAGAGGAGGAGGATGAAA	SEQ?ID?NO：233
HPV16E7?34	AGGATGAAATAGATGGTCC	SEQ?ID?NO：234
			HPV16E7?35	CCTACATTGCATGAATATA	SEQ?ID?NO：235
HPV16E7?36	ACAGAGCCCATTACAATAT	SEQ?ID?NO：236
			HPV16E7?37	ACACGTAGACATTCGTACT	SEQ?ID?NO：237
HPV16E7?38	GTACTTTGGAAGACCTGTT	SEQ?ID?NO：238
			HPV16E7?39	CCTGTTAATGGGCACACTA	SEQ?ID?NO：239
HPV16E7?40	CTACTGTTATGAGCAATTA	SEQ?ID?NO：240
			HPV16E7?41	ACAGCTCAGAGGAGGAGGA	SEQ?ID?NO：241
HPV16E7?42	GGAGATACACCTACATTGC	SEQ?ID?NO：242
			HPV16E7?43	ACATTCGTACTTTGGAAGA	SEQ?ID?NO：243
HPV16E7?44	TTAATGGGCACACTAGGAA	SEQ?ID?NO：244
			HPV16E7?45	TAATGGGCACACTAGGAAT	SEQ?ID?NO：245
HPV16E7?46	CAACCAGAGACAACTGATC	SEQ?ID?NO：246
			HPV16E7?47	ACGTAGACATTCGTACTTT	SEQ?ID?NO：247
HPV16E7?48	ACCTACATTGCATGAATAT	SEQ?ID?NO：248
			HPV16E7?49	GAGGAGGATGAAATAGATG	SEQ?ID?NO：249
HPV16E7?50	CAATTAAATGACAGCTCAG	SEQ?ID?NO：250
			HPV16E7?51	AGAACCGGACAGAGCCCAT	SEQ?ID?NO：251
HPV16E7?52	GCATGAATATATGTTAGAT	SEQ?ID?NO：252
			HPV16E7?53	CAGAGACAACTGATCTCTA	SEQ?ID?NO：253
HPV16E7?54	CAACTGATCTCTACTGTTA	SEQ?ID?NO：254
			HPV16E7?55	ACTGTTATGAGCAATTAAA	SEQ?ID?NO：255
HPV16E7?56	AGATGGTCCAGCTGGACAA	SEQ?ID?NO：256
			HPV16E7?57	GCCCATTACAATATTGTAA	SEQ?ID?NO：257
HPV16E7?58	TGTTGCAAGTGTGACTCTA	SEQ?ID?NO：258
			HPV16E7?59	GCTTCGGTTGTGCGTACAA	SEQ?ID?NO：259

[0054]?

HPV16E7?60	CGGTTGTGCGTACAAAGCA	SEQ?ID?NO：260
			HPV16E7?61	CAGAACCGGACAGAGCCCATTACAA	SEQ?ID?NO：261
HPV16E7?62	GAACCGGACAGAGCCCATTACAATA	SEQ?ID?NO：262
			HPV16E7?63	GGACAGAGCCCATTACAATATTGTA	SEQ?ID?NO：263
HPV16E7?64	GCGTACAAAGCACACACGTAGACAT	SEQ?ID?NO：264
			HPV16E7?65	CAAAGCACACACGTAGACATTCGTA	SEQ?ID?NO：265
HPV16E7?66	CACACACGTAGACATTCGTACTTTG	SEQ?ID?NO：266
			HPV16E7?67	CATTCGTACTTTGGAAGACCTGTTA	SEQ?ID?NO：267
HPV16E7?68	GGAAGACCTGTTAATGGGCACACTA	SEQ?ID?NO：268
			HPV16E7?69	GAAGACCTGTTAATGGGCACACTAG	SEQ?ID?NO：269
HPV16E7?70	GACCTGTTAATGGGCACACTAGGAA	SEQ?ID?NO：270

[0055]?

Embodiment two: the described dsRNA sequence of external checking suppresses the efficient that HPV E7 expresses

(1) cell cultures

(the SiHa of human cervical carcinoma cell system, ATCC) application contains penicillin-Streptomycin sulphate-Xin Meisu (PSN of 1%, Gibco) antibiotic mixture and 10% calf serum (FBS, Gibco) MEM substratum (Gibco) is cultivated in the cell culture incubator of 37 ℃, 5% carbonic acid gas and 95% atmospheric moisture.

(2) cell transfecting

(1) transfection the day before yesterday, the SiHa cell is not comprising antibiotic inoculation of medium, and the degree of converging of cell is 30～50% during to transfection.

(2) preparation transfection sample:

Be respectively siRNA (seeing Table 2) and the 0.5 μ l cell transfecting reagent Lipofectamine of 20 μ M with 1 μ l concentration ^TM2000 (Invitrogen) are diluted in 0.05ml Opti-MEM (Invitogen), and mixing was hatched 5 minutes gently.Mixing was hatched 5 minutes gently.

(its positive-sense strand comprises: 5-UUCUCCGAACGUGUCACGU-3 with meaningless contrast siRNA; Antisense strand comprises: 5-ACgUgACACgUUCggAgAA-3) with cell transfecting reagent Lipofectamine ^TM2000 (Invitrogen) carry out transfection, as normal control group (NC group).

Get siRNA group and the NC group of dilution, mix gently, at room temperature hatched 20 minutes, form siRNA-Lipofectamine2000 (Invitrogen) mixture and meaningless siRNA-Lipofectamine2000 (Invitrogen) mixture.Incubation time is no more than 30 minutes, and longer incubation time may reduce activity.

The siRNA-LipofectamineTM2000 mixture is joined in each hole that comprises cell and substratum, mix by the culture plate that rocks back and forth lightly, at 37 ℃, CO ₂Incubator was hatched 24～96 hours, up to being fit to carry out the gene disruption analysis.Do not need to remove mixture or change substratum.Yet, after transfection, changed the activity that substratum also can not lose transfection in 4～6 hours.

Table 3 uses preferred amount and the volume of reagent when having provided by various tissue culture mode transfectional cell.

Use amount and the volume of reagent during table 3 transfectional cell

(3) total RNA extraction, reverse transcription and real-time quantitative PCR detect genetic expression

Transfection 48 hours later, collecting cell.(RNeasy Mini Kit, QIAGEN Cat.No.74106) carry out the extracting of cell RNA to cultured cells with the RNA extraction agent box of QIAGEN company.The sucking-off cell culture fluid with containing 0.1～0.25% tryptic PBS flushing cell, adds the substratum that contains serum then and activates trypsinase, with cell transfer to the RNase-free centrifuge tube, centrifugal 5 minutes of 300 * g.After centrifugal, add 350 μ l RLT damping fluid dissolved cells, mix, centrifugal 5 minutes of 8000 * g (10000rpm), abandoning supernatant adds 70% volume of ethanol mixing, is transferred on the RNeasy column spinner film, centrifugal 15 seconds of 8000 * g (10000rpm), damping fluid RW1 flushing RNeasy column spinner film.Add RNase-free water, centrifugal 1 minute of 8000 * g (10000rpm) namely extracts and obtains total RNA.With spectrophotometric determination RNA purity.The absorption value of 1 unit is equivalent to 0.04 μ g/ μ l RNA.

The RNA reverse transcription of 1 μ g extracting is become cDNA.The RNA that extracts is blown afloat with DEPC water, add 50ng/ μ l random primer 1 μ l, add 1mM dNTP1 μ l, put into 65 ℃ of water-bath 5min, fast ice bath.Centrifugal, collecting precipitation adds 4 μ l5 * First-Stand damping fluid, 2 μ l0.1M DTT and 1 μ lRNaseOUT ^TM(40 units/μ l), mixing adds 1 μ l (200 unit) SuperScript ^TMIIRT.42 ℃ of water-baths 50 minutes, 70 ℃ of water-baths are 15 minutes then.

Be that template is carried out the expression level that real-time quantitative polymerase chain reaction (Real time PCR) comes detection by quantitative HPV16E7mRNA with this cDNA.At first adopt SDS2.1 software relative standard curve method to calculate the mRNA expression level, determine Ct value (Ct) from corresponding curve, Ct value reflects relative expression's level of each target gene.Then, obtain the normality target value by contrasting the division aim parameter with reference to gene (glyceraldehyde-3-phosphate dehydrogenase is abbreviated as GAPDH) in the cell, thereby determine relative expression's level of mRNA.

The primer sequence of using in the experiment:

HPV16E7 upstream primer: 5 '-CGGACAGAGCCCATTACAATATT-3 '

HPV16E7 downstream primer: 5 '-CGCACAACCGAAGCGTAGA-3 '

People GAPDH upstream primer: 5 '-GGCATGGACTGTGGTCATGAG-3 '

People GAPDH downstream primer: 5 '-TGCACCACCAACTGCTTAGC-3 '

Calculating the efficient of the siRNA of institute inhibition of gene expression can calculate by following formula:

" processing cell " refers to the cell of siRNA in the transfection table 1 respectively in the formula, and " control cells " refers to the cell of the meaningless contrast of transfection siRNA, and " relative expression's value " is the ratio of HPV E7mRNA and GAPDH mRNA in the phalangeal cell.

Table 4 shows the efficient that the inhibition HPV E7mRNA of transfection described siRNA after 48 hours expresses.

The inhibition efficient that the HPV E7mRNA of table 4siRNA expresses

Double-stranded title	Suppress efficient	Standard deviation
			HPV16E7si01	95.8％	2.3％
HPV16E7si02	93.1％	3.3％
			HPV16E7si03	97.5％	1.5％
HPV16E7si04	90.2％	3.4％
			HPV16E7si05	83.1％	3.2％
HPV16E7si06	88.2％	4.1％
			HPV16E7si07	60.3％	2.6％
HPV16E7si08	77.9％	6.3％
			HPV16E7si09	40.5％	9.3％
HPV16E7si10	91.4％	1.2％
			HPV16E7si11	84.2％	3.9％
HPV16E7si12	79.6％	8.4％
			HPV16E7si13	77.9％	5.3％
HPV16E7si14	86.6％	5.2％
			HPV16E7si15	91.5％	2.1％
HPV16E7si16	50.2％	10.4％
			HPV16E7si17	60.6％	4.3％
HPV16E7si18	79.3％	8.1％

[0090]?

HPV16E7si19	82.5％	7.5％
			HPV16E7si20	89.8％	3.0％
HPV16E7si21	70.1％	4.3％
			HPV16E7si22	82.0％	7.2％
HPV16E7si23	63.7％	7.9％
			HPV16E7si24	65.9％	5.4％
HPV16E7si25	91.5％	2.4％
			HPV16E7si26	76.7％	12.3％
HPV16E7si27	82.7％	8.6％
			HPV16E7si28	54.6％	7.5％
HPV16E7si29	90.1％	2.2％
			HPV16E7si30	77.2％	3.4％
HPV16E7si31	81.2％	7.1％
			HPV16E7si32	84.7％	3.6％
HPV16E7si33	68.9％	6.6％
			HPV16E7si34	73.3％	7.9％
HPV16E7si35	73.5％	4.3％
			HPV16E7si36	84.8％	7.2％
HPV16E7si37	78.2％	4.5％
			HPV16E7si38	79.3％	4.1％
HPV16E7si39	67.5％	7.8％
			HPV16E7si40	49.2％	8.6％
HPV16E7si41	78.4％	8.7％
			HPV16E7si42	87.6％	3.1％
HPV16E7si43	74.4％	9.8％
			HPV16E7si44	85.5％	3.6％
HPV16E7si45	91.0％	2.3％
			HPV16E7si46	53.9％	10.9％
HPV16E7si47	82.7％	4.7％
			HPV16E7si48	75.3％	5.1％

[0091]?

HPV16E7si49	77.7％	6.1％
			HPV16E7si50	84.5％	3.8％
HPV16E7si51	62.8％	7.5％
			HPV16E7si52	78.3％	7.2％
HPV16E7si53	74.5％	6.2％
			HPV16E7si54	86.2％	4.3％
HPV16E7si55	79.6％	5.1％
			HPV16E7si56	86.3％	5.8％
HPV16E7si57	77.1％	9.1％
			HPV16E7si58	89.5％	1.8％
HPV16E7si59	51.3％	7.8％
			HPV16E7si60	72.6％	7.9％
HPV16E7si61	96.3％	1.4％
			HPV16E7si62	92.5％	2.1％
HPV16E7si63	94.5％	3.6％
			HPV16E7si64	91.7％	2.5％
HPV16E7si65	85.6％	1.9％
			HPV16E7si66	87.3％	4.2％
HPV16E7si67	91.2％	2.2％
			HPV16E7si68	90.1％	2.1％
HPV16E7si69	80.9％	6.9％
			HPV16E7si70	77.6％	8.7％
HPV16E7si71	96.6％	4.2％
			HPV16E7si72	92.8％	7.6％
HPV16E7si73	87.4％	5.3％
			HPV16E7si74	91.2％	6.9％
HPV16E7si75	79.5％	6.2％
			HPV16E7si76	88.3％	7.9％
HPV16E7si77	95.7％	4.4％
			HPV16E7si78	79.6％	8.3％

[0092]?

HPV16E7si79	88.2％	6.4％
			HPV16E7si80	76.1％	8.5％
HPV16E7si01ome	93.7％	4.8％
			HPV16E7si02F	96.2％	6.7％
HPV16E7si03ome	94.4％	2.9％
			HPV16E7si04ome	84.3％	8.3％
HPV16E7si05ome	79.8％	4.6％
			HPV16E7si06S	88.7％	7.2％
HPV16E7si07S	72.5％	6.3％
			HPV16E7si08F	82.6％	5.7％
HPV16E7si09S	47.9％	5.2％
			HPV16E7si10ome	84.2％	7.3％

The inhibition of the albumen of three couples of E7 of embodiment

One, sample preparation

According to embodiment one described method transfectional cell centrifugal collecting cell after 72 hours, cell sample adds 50～100ul lysate (prescription: 50mM Tris-base, 1.0mM EDTA, 150mM NaCl, 0.1%SDS, 1%TritonX-100,1%sodium deoxycholate (sodium deoxycholate), 1%PMSF), suspend, the centrifugal 30min of ultrasonic back 20,000g gets supernatant.

Concentration waits sample qualitatively per sample.

Two, SDS-PAGE protein electrophorese

1, joins separation gel

I) offset plate cleans, the distilled water flushing, and the dehydrated alcohol dehydration, blower dries up puts on the shelf, and the transparent adhesive tape leakproof is sealed up in the glass lower edge;

Ii) add clamping plate, to the 1cm of broach lower end;

Add about 100ul propyl carbinol on the iii separation gel and push down, rock and make the glue face smooth;

Iv) behind about 90min, water and distilled water wash away propyl carbinol, and filter paper blots.

2, join concentrated glue

I) behind the adding clamping plate, insert comb (noting: bubble can not be arranged) immediately

3, electrophoresis

I) behind the concentrated about admittedly 10min of gelling, put into electrophoresis chamber, add the Gly electrophoretic buffer and check closure;

Ii) pull out broach, sample on the microsyringe (cumulative volume is less than 20ul);

Iii) make the periphery flood the electrophoresis platinum wire with damping fluid;

Iv) constant current 10mA/ piece glue is 60 minutes; 20mA/ piece glue 2 hours is advisable just to run out of glue;

After v) electrophoresis finishes, pry open offset plate;

Vi) remove upper strata glue, cut one jiao of marking on the Marker limit.

Three, change film

I) the fresh commentaries on classics film liquid of preparation, 4 ℃ of precoolings;

Ii) take off glue behind the SDS-PAGE electrophoresis, rinsing is five minutes in distilled water;

Iii) cut 2 of 1 of suitable big or small pvdf membrane and big slightly filter paper, pvdf membrane soaks into methyl alcohol;

Iv) filter paper, cotton, glue immerse changes 10-20min in the film liquid, (gum concentration hangs down many Jinhuis);

V) from negative electrode (one), put cotton, filter paper, glue, film, filter paper, cotton successively, to anode (+);

Vi) add ice chest, in basin ice, change film in 4 ℃ of refrigerators;

Vii) change film condition: 250mA, 2h.

Four, sealing

I) film is put into hybridization bag (facing up, down together), adds an amount of confining liquid (5% skimmed milk) liquid, prehybridization 1～2hr.

Five, primary antibodie is hatched

I) 1: 100 adding primary antibodie (mouse-anti people HPV16E7 monoclonal antibody is available from Abcam) in the confining liquid, hybridization is spent the night;

Ii) TBST prewashing 1min washes 3 times again, each 5min.

Six, two anti-hatching

I) adding in 1: 10,000 two anti-(sheep anti mouse is available from Abcam) in the confining liquid, hybridization 1h;

Ii) TBST prewashing 1min washes 3 times again, each 5min.

Seven, develop

I) add A, B colour developing liquid 1: 1, every cm2 adds 80ul, envelope film colour developing 5min

Ii) film places place's horse, X-ray sheet exposure 30s-5min

The 1min that iii) develops, clear water floats 30s, photographic fixing 1min, clear water floats 30s

Eight, gray analysis

Expression amount by HPV E7 albumen behind the Gel Pro Analyzer4.0 software analysis transfection siRNA after the film scanning, and calculate the jamming effectiveness of corresponding siRNA, the result is as shown in table 4.

Table 5 shows the efficient of transfection inhibition HPV E7 protein expression of described siRNA after 72 hours.

The inhibition efficient of the HPV E7 of table 5siRNA protein expression

Double-stranded title	Suppress efficient	Standard deviation
			HPV16E7si01	91.8％	1.5％
HPV16E7si02	85.2％	4.5％
			HPV16E7si03	92.6％	2.3％
HPV16E7si04	87.3％	4.4％
			HPV16E7si05	80.2％	3.6％
HPV16E7si06	80.1％	4.5％
			HPV16E7si07	50.3％	7.6％

[0140]?

HPV16E7si08	69.7％	5.3％
			HPV16E7si09	20.5％	10.8％
HPV16E7si10	87.1％	5.2％
			HPV16E7si11	80.2％	4.8％
HPV16E7si12	70.5％	7.5％
			HPV16E7si13	67.6％	6.8％
HPV16E7si14	81.5％	6.2％
			HPV16E7si15	88.5％	4.3％
HPV16E7si16	35.9％	11.6％
			HPV16E7si17	50.0％	6.1％
HPV16E7si18	71.2％	7.3％
			HPV16E7si19	77.6％	7.5％
HPV16E7si20	81.7％	3.8％
			HPV16E7si21	60.9％	4.9％
HPV16E7si22	75.2％	6.2％
			HPV16E7si23	49.6％	6.9％
HPV16E7si24	40.8％	3.4％
			HPV16E7si25	80.4％	7.7％
HPV16E7si26	68.5％	9.3％
			HPV16E7si27	73.8％	9.5％
HPV16E7si28	30.6％	8.8％
			HPV16E7si29	80.6％	5.2％
HPV16E7si30	60.2％	4.4％
			HPV16E7si31	68.5％	7.9％
HPV16E7si32	77.6％	4.7％
			HPV16E7si33	50.8％	10.7％
HPV16E7si34	54.2％	12.7％
			HPV16E7si35	60.7％	13.1％
HPV16E7si36	72.6％	6.4％
			HPV16E7si37	57.7％	9.5％

[0141]?

HPV16E7si38	59.0％	8.1％
			HPV16E7si39	43.5％	6.3％
HPV16E7si40	26.8％	13.8％
			HPV16E7si41	65.2％	8.5％
HPV16E7si42	69.7％	12.1％
			HPV16E7si43	57.7％	10.8％
HPV16E7si44	74.5％	8.5％
			HPV16E7si45	78.0％	7.3％
HPV16E7si46	41.2％	12.9％
			HPV16E7si47	78.4％	7.3％
HPV16E7si48	76.3％	12.2％
			HPV16E7si49	62.7％	7.3％
HPV16E7si50	75.2％	8.2％
			HPV16E7si51	32.8％	9.4％
HPV16E7si52	61.2％	10.2％
			HPV16E7si53	71.3％	9.1％
HPV16E7si54	60.5％	8.5％
			HPV16E7si55	70.3％	7.1％
HPV16E7si56	50.8％	18.9％
			HPV16E7si57	66.2％	9.2％
HPV16E7si58	80.2％	9.4％
			HPV16E7si59	49.3％	6.6％
HPV16E7si60	50.5％	10.7％
			HPV16E7si61	91.5％	3.1％
HPV16E7si62	87.8％	5.3％
			HPV16E7si63	89.5％	2.7％
HPV16E7si64	70.7％	9.7％
			HPV16E7si65	73.5％	7.6％
HPV16E7si66	80.3％	6.3％
			HPV16E7si67	78.6％	7.8％

[0142]?

HPV16E7si68	80.2％	5.1％
			HPV16E7si69	69.7％	7.9％
HPV16E7si70	50.8％	11.2％
			HPV16E7si71	83.6％	5.2％
HPV16E7si72	82.5％	6.7％
			HPV16E7si73	92.4％	6.8％
HPV16E7si74	86.4％	9.2％
			HPV16E7si75	66.7％	4.8％
HPV16E7si76	68.5％	8.9％
			HPV16E7si77	76.2％	7.4％
HPV16E7si78	69.6％	7.3％
			HPV16E7si79	90.3％	8.2％
HPV16E7si80	66.9％	5.8％
			HPV16E7si01ome	89.7％	8.4％
HPV16E7si02F	90.6％	7.3％
			HPV16E7si03ome	82.9％	7.9％
HPV16E7si04ome	75.8％	6.2％
			HPV16E7si05ome	63.3％	8.6％
HPV16E7si06S	91.7％	6.9％
			HPV16E7si07S	80.5％	8.3％
HPV16E7si08F	67.7％	6.8％
			HPV16E7si09S	50.3％	9.6％
HPV16E7si10ome	86.3％	8.1％

Embodiment four: the described siRNA sequence inhibition of experimental verification can be expressed the growth of the implantation cervical cancer cell of HPV16E7 in the mouse body

Transfection HPV16E7si01, HPV16E7si0, HPV16E7si03 and meaningless dsRNA cervical cancer cell (SiHa) be will distinguish and the subcutaneous lotus knurl in big C57BL/6 female mice back of 6 weeks, each injected in mice 2x10 will be expelled to ⁷Individual cell.Target gene mRNA expresses in the mouse body of the different dsRNA of 3 all post analysis the effect of mourning in silence and knurl body weight.Experimental result such as following table 6 show that the dsRNA of 3 kinds of external efficient inhibition HPV E7 expression also can efficiently suppress the expression of HPV E7 in the mouse body, and can suppress to implant the growth of knurl body.

The inhibition efficient of the mouse tumor growth of table 6siRNA

Claims (6)

1. the HPV16E7 gene small molecule interference nucleic acid target sequence of a separation, wherein said target sequence is the sequence shown in the SEQ ID NO:202 in the table 1.

2. small RNA molecule, it instructs the shearing of the HPV16E7mRNA sequence that exists in the cell, thereby realize the inhibition to the HPV16E7 expression, this small RNA molecule positive-sense strand is 5 '-GGACAGAGCCCAUUACAAUTT-3 ', and antisense strand is 5 '-AUUGUAAUGGGCUCUGUCCTT-3 '.

3. small RNA molecule according to claim 2, wherein at least one Nucleotide is the Nucleotide of chemically modified.

4. small RNA molecule according to claim 3, wherein said chemically modified are at least a in the following modification:

(1) to connecting the modification of the phosphodiester bond of Nucleotide in the nucleotide sequence of described small RNA molecule;

(2) to the modification of 2 '-OH of the ribose in the nucleotide sequence of described small RNA molecule;

(3) to the modification of the base in the nucleotide sequence of described small RNA molecule.

5. composition that comprises each described small RNA molecule of claim 2-4 and pharmaceutically acceptable carrier.

6. the described composition of claim 5 is characterized in that pharmaceutically acceptable carrier is liposome or high molecular polymer.