CN1869237A

CN1869237A - Construction method of bismall molecule interference RNA carrier and its application

Info

Publication number: CN1869237A
Application number: CNA2006100191432A
Authority: CN
Inventors: 吴建国; 邬开朗; 朱应
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2006-05-26
Filing date: 2006-05-26
Publication date: 2006-11-29

Abstract

The invention discloses a construction method and application for a double micro-molecule disturbance RNA carrier. It includes the following steps: compounding construction primer of double siRNA producing carrier, PCR expanding of siRNA producing module; cloning and sequencing. The invention has the advantages of simple method to construct siRNA carrier, convenient to operate, and high RNA disturbance efficiency. It could make the gene expression that has the same source sequence of siRNA dumbness and would not influence to other functions of the gene. It has strong specificity, and no poison function. It is a good method to cure virus mixed infection by the double siRNA.

Description

A kind of construction process of bismall molecule interference RNA carrier and application

Technical field

The present invention relates to biological technical field.Specifically, the present invention relates to a kind of pair of siRNA construction of carrier, the purposes of this carrier in preparation or prevention antiviral.

Background technology

70% of human communicable disease is to have virus to cause, the wide power of the strong propagation of viral infection is big, influenza, atypical pneumonia, measles, encephalitis B, hepatitis B, poliomyelitis, rabies, human beings'health in virus disease serious threats such as AIDS, influences the development of world economy.With hepatitis B virus and human immunodeficiency virus is example: hepatitis B virus (HBV) infects and can cause acute and the chronic hepatopathy change.The number of the liver cirrhosis that causes owing to hepatitis B virus infection or liver cancer is died from above 1 million people in the annual whole world.Reached 5 ten thousand people by the number of this virus infection every year.In the worldwide, nearly 2,000,000,000 people are by the hepatitis B virus infection mistake, and it is long-term carrier of disease of hepatitis B virus that 300,000,000 5 thousand ten thousand people are arranged.Chinese then 100,000,000 2 thousand ten thousand people are arranged is that (Liu Chongbai etc. (1997) Chinese public health is learned, 13:515) for the long-term carrier of virus.Human immunodeficiency virus (HIV) infects can cause AIDS.AIDS is referred to as century plague.Since found the first AIDS in 1981, the number that infect in the whole world reaches 6,000 ten thousand according to estimates, and the people who dies from AIDS has reached 1,400 ten thousand, and increase with 16000 number of the infected every day.And in the crowd that HIV infects, exist the phenomenon of polyinfections such as HBV or HCV very general.But the treatment of virus disease is world's difficult problem, does not have good medicine or method.The treatment of antiviral has nearly 30 years history, owing to after virus enters body, in host cell, duplicate and breed, therefore, the medicine that can suppress viral proliferation has in various degree infringement to host cell or body, makes the slower development of antiviral drug.Up to the present, still there is not the antiviral drug that makes us satisfied fully.Is the choice drug of the treatment chronic hepatitis B of generally acknowledging at present as the anti-hbv drug interferon-alpha, but its curative effect only can make 25%～40% case obtain effectively to answer; Lamivudine has obtained certainly to the restraining effect of HBV DNA and the short term efficacy of treatment HBV infection, but prolonged application can induce HBV to produce resistance.Zidovudine (zidovudine) is that first is used for the treatment of the medicine that HIV infects, but it is unsatisfactory to suppress virus replication, and causes the resistance of virus to occur fast.And the RNA perturbation technique that development in recent years is got up is suppressing efficient height on the virus replication, and high specificity does not have toxic side effect.Thereby the RNA interference has shown tempting prospect on antiviral therapy

(RNA interference RNAi) is meant the interior mRNA generation of the cell specificity degraded of endogenous or exogenous double-stranded RNA (dsRNA) mediation, thereby causes the target gene expression silence, the deficient phenomena of generation function corresponding phenotype in the RNA interference.RNAi is a kind of conservative defense mechanism that has from the unicellular lower eukaryote to the Mammals.Double-stranded RNA is cut into the small pieces RNA interfering (siRNA) of about 19-22nt by Dicer in cell, and then formation siRNA-albumen composition (siRNP), siRNP finally causes specific genetic expression silence (gene silencing) [Liu JY by the mRNA that identification, degraded have homologous sequence, Jia LT, Wang CJ, et al.RNAi-genesilencing mediated by dsRNAs.J.Section Genet Foreign Med Sci, 2004; 27 (1): 4-10.].The RNA perturbation technique is subject to people's attention in recent years day by day, has been widely applied to target checking in the determining of function, the cellular signal transduction pathways analysis of research gene, redundant gene, the drug development, gene therapy, virus infection, cancer and dominant hereditary disease etc.The virus of having studied aspect anti-virus infection has: as poliovirus, human immunodeficiency virus, beastly canopy virus, Rous sarcoma virus, dengue virus, hepatitis C virus, human papillomavirus, influenza virus, hepatitis B virus, sars coronavirus etc.But these are to suppress a certain gene of virus and reach duplicating of a kind of virus of inhibition with a kind of siRNA.And express two kinds of bismall molecule interference RNA carriers with identical carrier, and suppress the antiviral therapy of the polyinfection of two kinds of viruses, do not appear in the newspapers at present.

Summary of the invention

The objective of the invention is to be to provide a kind of bismall molecule interference RNA (siRNA) construction of carrier, method is simple, easy to operate, can make up two siRNA carriers with this method, this siRNA carrier silencing virus gene and reach and suppress duplicating of virus at any two kinds of viruses.

Another object of the present invention provides the application of a kind of couple of siRNA in preparing treatment or inhibition hepatitis B virus (HBV), human immunodeficiency virus (HIV) polyinfection medicament.Method with vector expression prepares siRNA, and method is simply with low cost.RNA jamming effectiveness height, the efficient of suppressor gene table can reach more than 90%, only make to have the function that does not influence other gene with the genetic expression silence of siRNA homologous sequence, so high specificity has no side effect.Therefore are a kind of very promising methods with two siRNA treatment virus mixed infections.

In order to achieve the above object, the present invention is by the following technical solutions:

1, the structure primer of two siRNA generation carriers is synthetic: designed a pair of primer, upstream primer:

5 '-GCTGATGACGTCAGTGGAAAGACGCG-3 ' downstream primer:

5’-TCAGCGAATTCACGCCAAGCTTTTCC-3’。This upstream primer contains one section sequence of U6 promotor and introduces the restriction endonuclease sites of AatII, downstream primer is one section sequence between the small molecular interfering RNA expression vector (HBssiRNA) last 788～807 of hepatitis B virus S gene, and introduces the restriction endonuclease sites of an EcoRI.Send Invitrogen company synthetic then.

2, small molecules interference RNA produces the template pcr amplification: the small molecular interfering RNA expression vector (HBssiRNA) with hepatitis B virus S gene is a template, and increasing with above-mentioned steps 1 synthetic primer obtains the PCR product.With 1% sepharose the PCR product is carried out electrophoresis, with the DNA purifying recovery test kit purifying of Qiagen company.

3, clone and order-checking: the dna fragmentation that reclaims is carried out being connected to behind the double digestion between the AatII and EcoRI site on human immunodeficiency virus's small molecular interfering RNA expression vector (HIVsiRNA) with AatII and EcoRI; produce one and can express the small molecules interference RNA of hepatitis B virus (HBs) S gene and the plasmid psilencer2.1-U6-HBV-HIVsiRNA of human immunodeficiency virus (HIV) two small molecules interference RNAs of small molecules interference RNA (siRNA) (being called for short HBV-HIVsiRNA) simultaneously, the positive colony that screens serves Hai Huanuo and order-checking confirms.(the acquisition of HBssiRNA and the HIVsiRNA document Kailang Wu that sees reference, Xue Zhang, Jianlin Zhang, Yongbo Yang, Yong-Xin Mu, Mo Liu, Lu Lu, Yan Li, Ying Zhu, Jianguo Wu.Inhibition of Hepatitis B Virus Gene Expression by Single and Dual SmallInterfering RNA Treatment.Virus Research 2005.Sept, 112 (1-2): 100-107.).Building process is seen Fig. 1.

Two siRNA carriers have been obtained by above step, this carrier is to the application in preparation or inhibition HBV polyinfection medicament, the application of this carrier in preparation or inhibition HIV polyinfection medicament simultaneously, this carrier to the inhibition application process of duplicating of HBV, HIV is:

A, two siRNA produce the inhibition test of duplicating of carrier to HBV:

1) preparation of transfection plasmid solution: the water that 1mg-10mg HBV-HIVsiRNA plasmid is dissolved in the 1mL sterilization.

2) transfection: get HepG2.2.15 cell on the tissue culture dish that cationic-liposome (Xiamen sun horse Bioisystech Co., Ltd) transfection that the above-mentioned solution of 1uL-10uL adds 5uL-10uL 5mg/mL is grown in 6 holes (and with the carrier of expressing irrelevant siRNA as negative control).

3) the hbv replication intermediate extracts: the HepG2.2.15 cell cultures that 6 orifice plates are cultivated was washed once with cold PBS (pH7.4) after 48 hours, 5) (composition of lysate is 50mM Tris-Cl to add the cell pyrolysis liquid in 300uL/ hole then, pH7.4,1mM EDTA, 1%Nonidet P-40) room temperature (20-25 ℃) is placed 15min～30min and is made its abundant cracking; Forward cell pyrolysis liquid in the 1.5mL EP pipe centrifugal 1min of 14000rpm; Get and reset and add 10mMMgCl and 100ug/mL DNase I and put 37 ℃ and handle 30min, adding EDTA again is the 25mM termination reaction to final concentration; Adding Proteinase K, to make its final concentration be 0.5ug/mL, and 37 ℃ of insulation 2hr are with digestible protein; Extracting once with phenol and chloroform mixed solution (both volume ratios are 1: 1); With dehydrated alcohol deposit D NA, the aqua sterilisa dissolving of dry back.

4) HBV suppresses efficiency test: the copy number of measuring HBV with fluorescent quantificationally PCR detecting kit (production of Shenzhen PG bio tech ltd).Virus replication inhibiting rate=(1-sample well copy number/control wells copy number) * 100% the results are shown in Table 1.

Table 1 couple siRNA is to the inhibition effect of HBV and HIV

	HBV	HIV
	HBV	HIV	Suppress (%)	83.5	78.3

Measure the copy number of the replicative intermediate of HBV with HBV-HIVsiRNA transfection HepG2.2.15 cell and then with quantitative fluorescent PCR, calculate inhibiting rate HBV.Detect the proteic concentration of p24 with the p24 protein detection kit then with HBV-HIVsiRNA and pNL4-3 cotransfection HEK293T, calculate inhibiting rate HIV.

B, two siRNA produce the inhibition test of duplicating of carrier to HIV:

1) preparation of transfection plasmid solution: the water that 1mg-10mg HBV-HIVsiRNA plasmid is dissolved in the 1mL sterilization.PNL4-3 (purchasing in NIH) is that the infectivity plasmid of HIV-1 can duplicate and produce infective virion in the HEK293T cell.Be made into the solution of 1mg-10mg/mL equally with aqua sterilisa.

2) transfection: get HEK293T cell on the tissue culture dish that cationic-liposome (Xiamen sun horse Bioisystech Co., Ltd) cotransfection that 1uL-5uLHBV-HIVsiRNA plasmid solution and 1uL-5uL pNL4-3 plasmid solution add 5uL-10uL 5mg/mL is grown in 6 holes (and with the carrier of expressing irrelevant siRNA as negative control).

3) HIV suppresses efficiency test: cell is cultivated after 72 hours again and is measured the proteic concentration of HIV-1 p24 in the cell culture supernatant with p24 protein detection kit (purchasing the company in Ghent Belgium) after the transfection.Inhibiting rate=(1-sample well concentration value/control wells concentration value) * 100%.The results are shown in Table 1.

Invention advantage and effect

The invention provides a kind of pair of siRNA carrier, this carrier can be expressed two kinds of siRNA and be suppressed duplicating of two kinds of viruses.The present invention also provides the method for a kind of pair of siRNA vector construction.Method is simple, and is easy to operate, with low cost.RNA jamming effectiveness height, the efficient of suppressor gene table can reach more than 90%, only make to have the function that does not influence other gene with the genetic expression silence of siRNA homologous sequence, so high specificity has no side effect.Therefore are a kind of very promising methods with two siRNA treatment virus mixed infections.Existingly suppress a kind of a certain gene of virus and reach to suppress duplicating of a kind of virus with antiviral also the resting on of RNA perturbation technique with siRNA.Biggest advantage of the present invention is to suppress duplicating of two kinds of viruses simultaneously, is the biotechnological formulation that the utmost point has potential clinical value.

Description of drawings

Fig. 1. two siRNA produce the structure synoptic diagram of carrier

Be cloned into Aat II and EcoRI site on another siRNA expression vector after with a pair of primer a carrier U6 promotor and siRNA expression template being increased out again, constitute a carrier of expressing 2 siRNA simultaneously.

Embodiment

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described 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 people such as normal experiment condition such as Sambrook, molecular cloning, laboratory manual (third edition) (New York:ColdSpring Harbor Laboratory Press, 2002) condition described in, or the condition of advising according to manufacturer.

1. couples of siRNA of embodiment produce the structure of carrier

1) the structure primer of two siRNA generation carriers is synthetic: designed a pair of primer, upstream primer:

5 '-GCTGATGACGTCAGTGGAAAGACGCG-3 ' downstream primer:

5’-TCAGCGAATTCACGCCAAGCTTTTCC-3’。This upstream primer contains one section sequence of U6 promotor and introduces the restriction endonuclease sites of AatII, and downstream primer is one section sequence between the HBssiRNA last 788～807, and introduces the restriction endonuclease sites of an EcoRI.Send Invitrogen company synthetic then.

2) siRNA produces the template pcr amplification: with HBssiRNA is template, and increasing with above-mentioned synthetic primer obtains the PCR product.With 1% sepharose the PCR product is carried out electrophoresis, with the DNA purifying recovery test kit purifying of Qiagen company.

3) clone and order-checking: the dna fragmentation AatII that reclaims and EcoRI are carried out being connected to behind the double digestion between the AatII and EcoRI site on the HIVsiRNA; produce a plasmid psilencer2.1-U6-HBV-HIVsiRNA (being called for short HBV-HIVsiRNA) that can express two siRNA of siRNA of HBs and HIV-1 simultaneously, the positive colony that screens serves Hai Huanuo and order-checking confirms.

2. couples of siRNA of embodiment produce the inhibition of duplicating of carrier to HBV

3) the hbv replication intermediate extracts: the HepG2.2.15 cell cultures that 6 orifice plates are cultivated was washed once with cold PBS (pH7.4) after 48 hours, (main component of lysate is 50mMT-Cl to add the cell pyrolysis liquid in 300uL/ hole then, pH7.4,1mM EDTA, 1%Nonidet P-40) room temperature (20-25 ℃) is placed 15min～30min and is made its abundant cracking; Forward in the 1.5mLEP pipe cell pyrolysis liquid to 14, the centrifugal 1min of 000rpm; Get and reset and add 10mMMgCl and 100ug/mL DNase I and put 37 ℃ and handle 30min, adding EDTA again is the 25mM termination reaction to final concentration; Adding Proteinase K, to make its final concentration be 0.5ug/mL, and 37 ℃ of insulation 2hr are with digestible protein; Extracting once with phenol and chloroform mixed solution (both volume ratios are 1: 1); With dehydrated alcohol deposit D NA, the aqua sterilisa dissolving of dry back.

3. couples of siRNA of embodiment produce the inhibition of duplicating of carrier to HIV

1) preparation of transfection plasmid solution: the water that 1mg-10mg HBV-HIVsiRNA plasmid is dissolved in the 1mL sterilization.PNL4-3 (purchasing in America NI H) pNL4-3 is the infectivity plasmid of HIV-1, can duplicate and produce infective virion in the HEK293T cell.Be made into the solution of 1mg-10mg/mL equally with aqua sterilisa.

3) HIV suppresses efficiency test: cell is cultivated after 72 hours again and is measured the proteic concentration of HIV-1p24 in the cell culture supernatant with p24 protein detection kit (purchasing the company in Ghent Belgium) after the transfection.Inhibiting rate=(1-sample well concentration value/control wells concentration value) * 100%.The results are shown in Table 1.

Claims

1, a kind of construction process of bismall molecule interference RNA carrier, it comprises the following steps:

The structure primer that A, bismall molecule interference RNA produce carrier synthesizes: designed a pair of primer, upstream primer: 5 '-GCTGATGACGTCAGTGGAAAGACGCG-3 ', downstream primer: 5 '-TCAGCGAATTCACGCCAAGCTTTTCC-3 ';

B, small molecules interference RNA produce the template pcr amplification: the small molecular interfering RNA expression vector with hepatitis B virus S gene is a template, increase with A step synthetic primer and to obtain the PCR product, with 1% sepharose the PCR product is carried out electrophoresis, purifying reclaims the test kit purifying;

C, clone and order-checking: the dna fragmentation that reclaims is carried out being connected to behind the double digestion between the AatII and EcoRI site on human immunodeficiency virus's small molecular interfering RNA expression vector with AatII and EcoRI, produce one and can express the small molecules interference RNA of hepatitis B virus S gene and the plasmid psilencer2.1-U6-HBV-HIVsiRNA of two small molecules interference RNAs of human immunodeficiency virus's small molecules interference RNA simultaneously, the positive colony order-checking that screens confirms, just obtains two siRNA expression vectors.

2, a kind of pair of application of siRNA carrier in preparation treatment or inhibition hepatitis B virus polyinfection medicament.

3, a kind of pair of application of siRNA carrier in preparation treatment or HIV inhibiting polyinfection medicament.