CN102925987A

CN102925987A - Construction method of random shRNA library

Info

Publication number: CN102925987A
Application number: CN2012104646926A
Authority: CN
Inventors: 杨国栋; 袁丽君; 王臻
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-11-19
Filing date: 2012-11-19
Publication date: 2013-02-13

Abstract

The invention discloses a construction method of a random shRNA library based on a pLKO.1 lentiviral vector. The method comprises the following steps: synthesizing a characterized nucleotide sequence (21 random nucleic acids being from 5' to 3'; a small stem-loop structure of a complementary chain being formed at the Xhol restriction enzyme cutting site), annealing the sequence and filling in to be a complete hairpin structure; adding a terminator sequence required by shRNA expression and 3'-terminated restriction enzyme cutting site EcoR1 required by cloning through a coupled reaction; on that basis, generating complementary chain through DNA polymerase and a single primer, connecting a restriction enzyme cutting site Agel to the lately generated chain 3'-terminal through coupled reaction; amplifying the sequences containing Agel and EcoR1 through a PCR (polymerase chain reaction) method; then, connecting PCR product to pMD-18 simple T carrier; then, removing extra sequences inside shRNA by an enzyme self ligation method, and at last subcloning insertion elements into pLKO.1 carrier to construct the shRNA library with entirely random expressing and packing into a virus. The library can be widely applied to screening disease-causing genes and drug targets.

Description

The construction process in a kind of at random shRNA library

Affiliated technical field

The present invention relates to life science, is a kind of strategy and method for making up shRNA library at random, and the application in drug target screening and critical function gene discovery and evaluation.

Background technology

RNAi is a kind of important biological phenomena of discovered in recent years, can characteristic reduces the amount of certain target gene interested in body and the cell.Based on such principle, scientists is by design shRNA, transfectional cell, and target is interfered candidate gene, for the understanding biological phenomena provides important means.On this basis, further design is for complete genomic RNA library, and the aspects such as the screening of finding and identify new functional gene, new drug target in full genome range, gene therapy are had broad application prospects.Conventional construction shRNA is by will then they being combined (Fig. 1) for heterogeneic sequence insertion vector one by one, forming the library.Progress along with the gene sequencing technology, new functional gene is constantly found in the full genome range, particularly some non-protein coding genes are found gradually, for human knowledge's biological phenomena and exploitation disease treatment means provide new candidate molecules, also had higher requirement in the shRNA library simultaneously.Then traditional shRNA library is mainly for known protein coding gene, and storage capacity is limited; Though some at random trials in shRNA library are arranged, these construction processs are introduced the problems such as extra sequence interference efficiency reduces, supporting virus vector virus packaging efficiency is lower because existing, and are unsatisfactory.Therefore the at random shRNA library that makes up based on high-level efficiency pLKO.1 carrier, satisfies fully the interference demand will promote the research in this field.

Summary of the invention

The technical problem that the present invention solves provides a kind of method completely random, that meet the double-stranded DNA of shRNA expression and machining feature that makes up.Another technical problem that the present invention solves provides the method that above-mentioned double-stranded DNA is cloned into pLKO.1 and efficiently expresses shRNA.

The beneficial effect of patent of the present invention is, the genes involved that participates in concrete biological function for screening provides reliable and stable instrument, for discovery and the checking of drug target provides effective means, and then promotes new drug development.

Description of drawings

Below in conjunction with accompanying drawing patent of the present invention is further specified: accompanying drawing 1 is the method for traditional structure pLKO.1 carrier, and prior art can not realize the in batches synthetic nucleotide sequence that can be transcribed into hairpin structure at random.Accompanying drawing 2-6 describes the method in the Random library that the present invention adopts, the method by fill (Fig. 2), connect add stop code and be connected ' restriction enzyme site 1 (Fig. 3), synthetic complementary strand, continue to connect and add the steps realizations such as 5 ' end restriction enzyme site (Fig. 4), amplification, clone (Fig. 5) and subclone (Fig. 6) and express the at random vector construction in shRNA library.

Embodiment

(1) by company's composition sequence, it is characterized in that:

5 '-NNNNNNNNNNNNNNNNNNNNNCTCGAGATCACCATTATAAGATCTCGAG-3 '; 3 ' end of this sequence can form a loop-stem structure, and wherein the structure of stem mainly is to realize by the restriction enzyme site of two Xho1;

(2) above-mentioned composition sequence is carried out 95 degree sex change 4min, then slow cooling to 20 degree makes it form incomplete hairpin structure (Fig. 2);

(3) in the nucleic acid solution of above-mentioned incomplete hairpin structure, add DNA polymerase Klenow fragment, buffer, dNTP, 25 degree 2 hours are finished extension, make it generate hairpin structure complete complementary, complete (Fig. 2);

(4) reclaim the DNA of above-mentioned hairpin structure by atmosphere/chloroform/ethanol precipitator method, be dissolved in H2O, add simultaneously the T4DNA ligase enzyme, connect damping fluid, with connexon linker1 pre-synthesis, 5 ' phosphorylation, its sequence signature is: 5 '-TTTTTAAGCTTCtcagcagagaactca-3 '; 16 degree connect and to spend the night, and make the DNA of hairpin structure possess termination signal and 3 ' the restriction enzyme site EcoR1 (Fig. 3) of 3 class promoter transcriptions;

(5) reclaim above-mentioned product, method is the same, on this basis, adds primer1, enzyme and the buffer of PCR reaction, and 95 degree sex change 3min, 74 degree are annealed and are extended 1.5min, generate complementary DNA (Fig. 3); The sequence of primer primer1 is: 5 '-TGAGTTCTCTGCTGAGAAGCTT-3 ';

(6) further reclaim the substrate that generates, 3 ' of complementary DNA is held carried out ligation; Its reaction process is with step 4, and the sequence of concrete linker2 is: 5 '-ACCGGTCCaccaactatccagac-3 '; The complementary DNA that the connection product is has possessed the restriction enzyme site (Fig. 3) of 5 ' end;

(7) reclaim above-mentioned connection product, add simultaneously primer 1 and primer2, wherein primer1 and 2 has all carried out 5 ' end and has added phosphatase reaction.The enzyme and dNTP, the buffer etc. that add the PCR reaction.The PCR reaction conditions is: 95 degree sex change 3min, and 74 degree annealing and extension 1min, 10 circulations (Fig. 4) are carried out altogether in reaction;

(8) reclaim above-mentioned PCR product, and be cloned into pMD-18 T simple carrier (ratio of carrier and fragment is 1: 1); This carrier does not contain the restriction enzyme sites such as Xho1, Age1 and EcoR1.Linked system is the same, and ligation is T4 DNA liagase16 degree effect 16 hours (Fig. 5);

(9) will connect product and be transformed into (during conversion, every 20ng carrier transforms 200 μ L competent cells) among the high efficiency DH5 α E.Coli, to guarantee obtaining maximum transfection efficiency, the bacterium after the conversion directly carries out microbial culture; In the microbial culture process, per 200 μ L competent cells are cultivated in the 200mL substratum;

(10) extract plasmid, cut by the Xho1 enzyme, glue directly connects after reclaiming carrier, so that the extra sequences such as loop are removed (Fig. 5);

(11) connect product and be transformed among the high efficiency DH5 α E.Coli the same step of process (9); Again extracting plasmid is connected into pLKO.1 after cutting by Age1 and EcoR1 enzyme, thereby successfully makes up the at random shRNA library (Fig. 6) that pLKO.1 expresses, and this library plasmid and VSVG, Δ 8.9 plasmid co-transfection HEK293T cells can be produced virus.Can obtain to express the virion of shRNA random library by the expansion scale.

Claims

1. construction process in shRNA library at random, it is characterized in that: (be followed successively by 21 any nucleic acid from 5 ' to 3 ' by the composite character nucleotide sequence, formed the little loop-stem structure of complementary strand by the Xho1 restriction enzyme site), by polymerization, ligation forms the double-stranded DNA structure of the shRNA that contains restriction enzyme site and completely random, be cloned into the T carrier, certainly the method that connects after cutting with the Xho1 enzyme again, remove the inner extra sequence of shRNA, cut back to close Insert Fragment in the T carrier by Age1 and EcoR1 enzyme at last, subclone to the pLKO.1 carrier, the pLKO.1 plasmid library of the shRNA of construction expression completely random.

2. according to the little loop-stem structure in the right characteristic nucleotide sequence 1 statement, synthetic, it is characterized in that: 5TCGAGATCACCATTATAAGATCTCGAG-3 '.

3. according to the Linker sequence of right 1 statement, its feature is respectively: connexon 1 sequence:

5 '-TTTTTAAGCTTCTCAGCAGAGAACTCA-3 '; Connexon 2 sequences:

5’-ACCGGTCCACCAACTATCCAGAC-3’。