CN111534520A

CN111534520A - Construction and application of lentivirus and recombinant vector for specifically inhibiting K-ras gene expression

Info

Publication number: CN111534520A
Application number: CN202010458555.6A
Authority: CN
Inventors: 徐新云; 郑凯; 毛吉炎; 蔡颖; 秦逍云; 秦双建; 李柏茹
Original assignee: Shenzhen Center For Disease Control And Prevention (shenzhen Health Inspection Center Shenzhen Institute Of Preventive Medicine)
Current assignee: Shenzhen Center For Disease Control And Prevention (shenzhen Health Inspection Center Shenzhen Institute Of Preventive Medicine)
Priority date: 2020-05-27
Filing date: 2020-05-27
Publication date: 2020-08-14

Abstract

The invention relates to construction and application of a lentivirus and a recombinant vector for specifically inhibiting K-ras gene expression. The lentivirus for specifically inhibiting the K-ras gene expression can express RNAi molecules for specifically inhibiting the K-ras gene expression, the RNAi molecules for specifically inhibiting the K-ras gene expression comprise a double-stranded RNA structure domain formed by an antisense chain and a sense chain which is hybridized with the antisense chain and consists of nucleotide sequences represented by SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, and the antisense chain is hybridized with the sense chain under strict hybridization conditions. The RNAi molecule for specifically inhibiting the K-ras gene expression can continuously, stably, efficiently and specifically inhibit the K-ras gene expression in human cells.

Description

Construction and application of lentivirus and recombinant vector for specifically inhibiting K-ras gene expression

Technical Field

The invention relates to the technical field of biology, in particular to construction and application of a lentivirus and a recombinant vector for specifically inhibiting K-ras gene expression.

Background

The K-ras gene is a protooncogene, about 35kb in length, located on chromosome 12, and is a member of the ras gene family, and K-ras-encoded K-ras protein has GTPase activity, is activated when binding GTP, and is inactivated when binding GDP. K-ras protein is mainly localized on cell membranes, and Protein Kinase C (PKC) can phosphorylate K-ras protein, and the phosphorylation process results in that the binding of K-ras protein and cell membranes is weakened to change the position, and the K-ras protein moves to endoplasmic reticulum, Golgi apparatus, mitochondria and other positions. The K-ras protein has molecular switching effect and is related to the generation, proliferation, migration, diffusion and angiogenesis of tumors.

In recent years, the advent of RNA interference (RNAi) has made it possible to regulate gene expression at the transcriptional level to specifically inhibit gene expression. Although RNA interference has application in the regulation of K-ras, the problems of unobvious interference effect and long-term stable interference are existed.

Disclosure of Invention

Based on the above, there is a need for an RNAi molecule capable of specifically inhibiting K-ras gene expression, which can persistently, stably, efficiently and specifically inhibit K-ras gene expression.

An RNAi molecule specifically inhibiting K-ras gene expression comprising a double-stranded RNA domain consisting of an antisense strand and a sense strand consisting of the nucleotide sequence represented by SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5 hybridized to said antisense strand, said antisense strand being hybridized to said sense strand under stringent hybridization conditions.

The shRNA for specifically inhibiting the K-ras gene expression can continuously, stably, efficiently and specifically inhibit the K-ras gene expression.

In one embodiment, the nucleotide sequence of the sense strand of the RNAi molecule is shown in SEQ ID No.1, and the nucleotide sequence of the antisense strand of the RNAi molecule is shown in SEQ ID No. 2;

or, the nucleotide sequence of the sense strand of the RNAi molecule is shown as SEQ ID NO.3, and the nucleotide sequence of the antisense strand of the RNAi molecule is shown as SEQ ID NO. 4;

or, the nucleotide sequence of the sense strand of the RNAi molecule is shown as SEQ ID NO.5, and the nucleotide sequence of the antisense strand of the RNAi molecule is shown as SEQ ID NO. 6.

In one embodiment, the sense strand of the RNAi molecule is linked to the antisense strand of the RNAi molecule by a stem-loop structure.

A recombinant vector for specifically inhibiting K-ras gene expression comprises a vector and a core fragment inserted in the vector, wherein the core fragment comprises a target sequence, a stem-loop structure sequence, a complementary sequence of the target sequence and a termination site which are sequentially connected, and the nucleotide sequence of the target sequence is shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5.

In one embodiment, the vector is a lentiviral vector; and/or the core fragment further comprises enzyme cutting sites, and the enzyme cutting sites are positioned at two ends of the core fragment.

In one embodiment, the nucleotide sequence of the sense strand of the core fragment is shown as SEQ ID No.7, and the nucleotide sequence of the antisense strand of the core fragment is shown as SEQ ID No. 8;

or, the nucleotide sequence of the sense strand of the core fragment is shown as SEQ ID No.9, and the nucleotide sequence of the antisense strand of the core fragment is shown as SEQ ID No. 10;

or, the nucleotide sequence of the sense strand of the core fragment is shown as SEQ ID No.11, and the nucleotide sequence of the antisense strand of the core fragment is shown as SEQ ID No. 12.

A construction method of a recombinant vector for specifically inhibiting K-ras gene expression comprises the following steps:

providing a core fragment, wherein the core fragment comprises a target sequence, a stem-loop structure sequence, a complementary sequence of the target sequence and a termination site which are connected in sequence, and the nucleotide sequence of the target sequence is shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5; and

and inserting the core fragment into a vector to prepare the recombinant vector for specifically inhibiting the expression of the K-ras gene.

A recombinant engineering bacterium, which contains the RNAi molecule for specifically inhibiting the expression of the K-ras gene;

or the recombinant engineering bacteria contain the recombinant vector for specifically inhibiting the K-ras gene expression.

A lentivirus for specifically inhibiting K-ras gene expression is prepared by the following steps:

inserting a core segment into a lentivirus and then transfecting a host cell, wherein the core segment comprises a target sequence, a stem-loop structure sequence, a complementary sequence of the target sequence and a termination site which are sequentially connected, and the nucleotide sequence of the target sequence is shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5; and

and amplifying the transfected host cells to obtain the lentivirus for specifically inhibiting the expression of the K-ras gene.

The RNAi molecule for specifically inhibiting the K-ras gene expression, the recombinant vector for specifically inhibiting the K-ras gene expression, the recombinant engineering bacteria or the lentivirus for specifically inhibiting the K-ras gene expression are applied to the preparation of medicines for treating diseases related to the abnormal K-ras gene expression.

A pharmaceutical composition, which comprises the RNAi molecule for specifically inhibiting the expression of the K-ras gene, the recombinant vector for specifically inhibiting the expression of the K-ras gene, the recombinant engineering bacteria or the lentivirus for specifically inhibiting the expression of the K-ras gene.

Drawings

FIG. 1 is a diagram showing the double-restriction enzyme electrophoresis of the recombinant vector in example 1;

FIG. 2 is a diagram showing a partial sequencing result of the pLVX-K-ras-shRNA1 recombinant vector in example 1;

FIG. 3 is a diagram showing a partial sequencing result of the pLVX-K-ras-shRNA2 recombinant vector in example 1;

FIG. 4 is a diagram showing a partial sequencing result of the pLVX-K-ras-shRNA3 recombinant vector in example 1;

FIG. 5 is a partial sequencing result diagram of the pLVX-shRNAC recombinant vector in example 1;

FIG. 6 shows the mRNA expression of K-ras corresponding to HBE cells transfected with four viruses in example 2;

FIG. 7 is the qPCR results of example 3;

FIG. 8 is a diagram showing the result of the Western blot detection electrophoresis in example 3;

FIG. 9 is a graph showing the quantitative results of Western blot detection in example 3;

FIGS. 10-15 are graphs showing the results of the effect of PM2.5 on the expression of k-ras, c-fos, c-myc, p53, Caspase-3 and Caspase-8 in this order.

Detailed Description

The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

One embodiment of the invention provides an RNAi molecule for specifically inhibiting K-ras gene expression. The RNAi molecule for specifically inhibiting the K-ras gene expression comprises a double-stranded RNA structure domain consisting of an antisense strand and a sense strand hybridized with the antisense strand and consisting of nucleotide sequences represented by SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.5, wherein the antisense strand is hybridized with the sense strand under strict hybridization conditions. The double-stranded RNA domain can target a specific sequence of the K-ras gene and inhibit the expression of the K-ras gene.

Specifically, the nucleotide sequence shown as SEQ ID NO.1 is: 5'-GGACGAATATGATCCAACA-3' are provided. Correspondingly, in an alternative embodiment, the nucleotides of the antisense strand of the RNAi molecule that specifically inhibits K-ras gene expression are shown in SEQ ID No. 2. The nucleotide sequence shown as SEQ ID NO.2 is as follows: 5'-TGTTGGATCATATTCGTCC-3' are provided.

Specifically, the nucleotide sequence shown as SEQ ID NO.3 is: 5'-GTGCAATGAGGGACCAGTA-3' are provided. Correspondingly, in an alternative embodiment, the nucleotides of the antisense strand of the RNAi molecule that specifically inhibits K-ras gene expression are shown in SEQ ID No. 4. The nucleotide sequence shown as SEQ ID NO.4 is: 5'-TACTGGTCCCTCATTGCAC-3' are provided.

Specifically, the nucleotide sequence shown as SEQ ID NO.5 is: 5'-GCTCTGAAGATGTACCTATG-3' are provided. Correspondingly, in an alternative embodiment, the nucleotides of the antisense strand of the RNAi molecule that specifically inhibits K-ras gene expression are shown in SEQ ID No. 6. The nucleotide sequence shown as SEQ ID NO.6 is: 5'-CATAGGTACATCTTCAGAGC-3' are provided.

In one embodiment, the sense strand of the RNAi molecule is linked to the antisense strand of the RNAi molecule by a stem-loop structure. In an alternative specific example, the nucleotide sequence of the stem-loop structure is shown in SEQ ID No.13, i.e.: 5 '-TTCAAGAGA-3'. Of course, in other embodiments, the nucleotide sequence of the stem-loop structure is not limited to the above, but may be other sequences commonly used in the art. Of course, in some embodiments, the stem-loop structure may also be omitted.

Note that "stringent conditions" as used herein are well known and include, for example, hybridization at 60 ℃ for 12 to 16 hours in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH6.4) and 1mM EDTA, followed by washing with a washing solution containing 0.1% SDS and 0.1% SSC at 65 ℃ for 15 to 60 minutes.

Proved by verification, the RNAi molecule for specifically inhibiting the K-ras gene expression can continuously, stably, efficiently and specifically inhibit the K-ras gene expression.

The invention also provides a recombinant vector for specifically inhibiting K-ras gene expression, which comprises a vector and a core fragment inserted in the vector, wherein the core fragment comprises a target sequence, a stem-loop structure sequence, a complementary sequence of the target sequence and a termination site which are sequentially connected, and the nucleotide sequence of the target sequence is shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5. The complementary sequence of the target sequence in the recombinant vector for specifically inhibiting the K-ras gene expression can target the specific sequence of the K-ras gene, so that the K-ras gene expression is inhibited.

Specifically, the nucleotide sequence of the termination site is shown as SEQ ID No.14, namely: 5 '-TTTTTT-3'. The specific nucleotide sequence shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5 is described above and is not described herein again. The nucleotide sequence of the stem-loop structure is as described above and will not be described in detail here.

In one embodiment, the core fragment further comprises a cleavage site located at each end of the core fragment to facilitate insertion of the core fragment into the vector. Furthermore, the enzyme cutting sites are BamH I enzyme cutting sites and EcoR I enzyme cutting sites. Of course, in other embodiments, the cleavage site is not limited to the above, and the cleavage site of the core fragment may be designed according to the cleavage site of the vector to be inserted. Of course, it is understood that in other embodiments, the cleavage site of the core fragment may be replaced by a cohesive end of the cleavage site. In this case, the step of cleaving shRNA that specifically inhibits the expression of the K-ras gene with an enzyme may be omitted when the core fragment is inserted into the vector.

In an alternative embodiment, the sense strand of the core fragment is shown as SEQ ID No.7 and the antisense strand of the core fragment is shown as SEQ ID No. 8. The nucleotide sequence shown as SEQ ID No.7 is as follows: 5'-GATCCGGACGAATATGATCCAACATTCAAGAGATGTTGGATCATATTCGTCCTTTTTT-3', respectively; the nucleotide sequence shown as SEQ ID No.8 is as follows: 5'-AATTAAAAAAGGACGAATATGATCCAACATCTCTTGAATGTTGGATCATATTCGTCCG-3' are provided.

In an alternative embodiment, the sense strand of the core fragment is shown as SEQ ID No.9 and the antisense strand of the core fragment is shown as SEQ ID No. 10. The nucleotide sequence shown as SEQ ID No.9 is as follows: 5'-GATCCGTGCAATGAGGGACCAGTATTCAAGAGATACTGGTCCCTCATTGCACTTTTTT-3', respectively; the nucleotide sequence shown as SEQ ID NO.10 is: 5'-AATTAAAAAAGTGCAATGAGGGACCAGTATCTCTTGAATACTGGTCCCTCATTGCACG-3' are provided.

In an alternative embodiment, the sense strand of the core fragment is shown as SEQ ID No.11 and the antisense strand of the core fragment is shown as SEQ ID No. 12. The nucleotide sequence shown as SEQ ID No.11 is as follows: 5'-GATCCGCTCTGAAGATGTACCTATGTTCAAGAGACATAGGTACATCTTCAGAGTTTTTT-3', respectively; the nucleotide sequence shown as SEQ ID No.12 is as follows: 5'-AATTAAAAAACTCTGAAGATGTACCTATGTCTCTTGAACATAGGTACATCTTCAGAGCG-3' are provided.

In this embodiment, the vector is a lentiviral vector. Specifically, the lentiviral vector comprises a basic sequence, a resistance gene sequence, a multiple cloning site sequence and a promoter sequence. In an alternative embodiment, the lentiviral vector is selected from the group consisting of pLVX-shRNA1, pLVX-shRNA2, and PLKO.1-PURO. Of course, in other embodiments, the vector is not limited to lentiviruses, but may be other vectors commonly used in the art.

The embodiment of the invention also provides a construction method of the recombinant vector for specifically inhibiting the K-ras gene expression, which comprises the following steps of S110 to S120:

and step S110, providing any one of the core fragments.

Specifically, the sense strand and the antisense strand of the core fragment are synthesized, and then the sense strand and the antisense strand are mixed and annealed to form the core fragment. Of course, the sense and antisense strands of the core fragment can also be obtained directly by means of gene synthesis.

And S120, inserting the core fragment into a vector to prepare a recombinant vector for specifically inhibiting the K-ras gene expression.

In particular, the choice of the carrier is as described above and will not be described in further detail here.

Specifically, the core fragment and the vector are subjected to double enzyme digestion treatment by using restriction enzymes respectively, and then the core fragment subjected to enzyme digestion treatment is inserted into the vector subjected to enzyme digestion treatment to obtain the recombinant vector for specifically inhibiting the K-ras gene expression.

In an alternative specific example, the vector is a lentiviral vector. Furthermore, the lentiviral vector has a BamHI cleavage site and an EcoRI cleavage site, and the core fragment also has a BamHI cleavage site and an EcoRI cleavage site. At the moment, carrying out double enzyme digestion treatment on the lentiviral vector and the core fragment by using a restriction enzyme BamHI and a restriction enzyme EcoRI, and opening a notch to form a viscous tail end of an enzyme digestion site of the lentiviral vector and a viscous tail end of the core fragment; then the core fragment is connected with the cohesive end of the enzyme cutting site of the lentivirus vector, thereby obtaining the recombinant vector for specifically inhibiting the K-ras gene expression.

Specifically, after a lentiviral vector and a core fragment are subjected to double enzyme digestion by using a restriction enzyme BamHI and a restriction enzyme EcoRI, the double-enzyme-digested core fragment is connected to the lentiviral vector subjected to double enzyme digestion by using a DNA ligase, and then the recombinant vector for specifically inhibiting the K-ras gene expression is obtained through amplification and identification. Further, the DNA ligase is T4 DNA ligase. The specific amplification and identification method may be any method commonly used in the art.

The construction method of the recombinant vector for specifically inhibiting the K-ras gene expression is simple to operate, and the recombinant vector capable of specifically inhibiting the K-ras gene is successfully constructed by inserting the core segment containing the target sequence into the vector. Experimental results show that the recombinant vector for specifically inhibiting the K-ras gene expression can be transfected into human cells, has high transfection efficiency and small dosage of the expression vector, and can continuously, stably, efficiently and specifically inhibit the K-ras gene expression in the human cells.

The invention also provides a lentivirus for specifically inhibiting K-ras gene expression, which is prepared by the following steps: after any core fragment is inserted into lentivirus and transfected into host cells, the transfected host cells are amplified to obtain the lentivirus for specifically inhibiting the K-ras gene expression.

In an alternative embodiment, the host cell is a 293T cell. Furthermore, the culture time of the 293FT cells after transfection is 24-48 h. Of course, in other embodiments, the host cell is not limited to 293T cells, but may be other host cells commonly used in the art.

The lentivirus capable of specifically inhibiting the K-ras gene expression can be transfected into a human cell, and the lentivirus can continuously, stably and efficiently inhibit the K-ras gene expression in the human cell.

An embodiment of the present invention also provides a cell in which expression of a K-ras gene is inhibited, which is prepared by the steps of: HBE cells (human bronchial epithelial-like cells) infected with the lentivirus specifically inhibiting the expression of the K-ras gene are used, and the infected HBE cells are cultured to obtain cells with the K-ras gene expression inhibited.

Specifically, HBE cells are confluent to 60-90% of cells, and the slow virus for specifically inhibiting K-ras gene expression is used for infecting the HBE cells, and the HBE cells are cultured for 24-48 hours at 36.5-38 ℃ to obtain the cells with the K-ras gene expression being inhibited.

The K-ras gene expression in the cells with the K-ras gene expression inhibition is continuously, stably and efficiently inhibited, and can be used as a cell model for K-ras gene related research.

The invention also provides a recombinant engineering bacterium, which contains any RNAi molecule for specifically inhibiting the expression of the K-ras gene or any recombinant vector for specifically inhibiting the expression of the K-ras gene.

The recombinant engineering bacteria contain any shRNA for specifically inhibiting the K-ras gene expression or any recombinant vector for specifically inhibiting the K-ras gene expression, and the K-ras gene expression is continuously, stably and efficiently inhibited.

The invention also provides an RNAi molecule for specifically inhibiting the expression of the K-ras gene, a recombinant vector for specifically inhibiting the expression of the K-ras gene, a recombinant engineering bacterium, a cell with the K-ras gene expression inhibited or an application of a lentivirus for specifically inhibiting the K-ras gene expression in preparing a medicament for treating diseases related to the abnormal expression of the K-ras gene.

The invention also provides a pharmaceutical composition, which comprises the RNAi molecule for specifically inhibiting the expression of the K-ras gene, the recombinant vector for specifically inhibiting the expression of the K-ras gene, the recombinant engineering bacteria, the cells with the K-ras gene expression inhibited or the lentivirus for inhibiting the K-ras gene expression.

In an alternative embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. The examples, which are not specifically illustrated, employ drugs and equipment, all of which are conventional in the art. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.

Example 1

Construction of vectors for silencing K-ras Gene

(1) Obtaining a core segment containing shRNA for specifically inhibiting K-ras gene expression

Sense and antisense strands of four core fragments were synthesized by Compton Biotechnology engineering (Shanghai) Inc. The four core fragments are: K-ras-shRNA1, K-ras-shRNA2, K-ras-shRNA3 and shRNAC, wherein the shRNAC is a control group, and the K-ras-shRNA1 contains a target sequence shown in SEQ ID No. 1; the K-ras-shRNA2 contains a target sequence shown as SEQ ID No. 3; the K-ras-shRNA3 contains a target sequence shown as SEQ ID No. 5. Wherein, a sense strand (K-ras-shRNA-T1) of K-ras-shRNA1 is shown as SEQ ID No.7, an antisense strand (K-ras-shRNA-D1) of K-ras-shRNA1 is shown as SEQ ID No.8, a sense strand (K-ras-shRNA-T2) of K-ras-shRNA2 is shown as SEQ ID No.9, an antisense strand (K-ras-shRNA-D2) of K-ras-shRNA2 is shown as SEQ ID No.10, a sense strand (K-ras-shRNA-T3) of K-ras-shRNA3 is shown as SEQ ID No.11, an shRNA strand (K-ras-shRNA-D3) of K-ras-shRNA3 is shown as SEQ ID No.12, a sense strand (shRNAC-TC) of shAC is shown as SEQ ID No.15, and an antisense strand (shAC-RNAC) of shAC 16 is shown as SEQ ID No. 16. The specific sequence is shown in Table 1.

TABLE 1

(2) The vector pLVX-shRNA1 is cut in two times: the pLVX-shRNA1 was digested simultaneously with EcoR1 and BamH1 at 37 ℃ for 30min, and then the restriction enzyme was added again to the mixture and digested at 37 ℃ for 30 min. The enzyme digestion reaction system is shown in the following table 2:

TABLE 2

The digested vector was recovered by agarose Gel electrophoresis, the details of which are described in the Gel extraction kit (D2500-01) of OMEGA.

(3) The double strands of the core fragment in (1) were mixed uniformly as shown in Table 3, and then placed in boiling water, allowed to cool naturally to room temperature (26 ℃ C.), and annealed. Then adding 1ml ddH into the core fragment after the annealing is finished₂O。

TABLE 3

(4) Construction of recombinant expression vectors: and (3) respectively connecting each core fragment with a cohesive end formed by annealing in the step (3) with the digested pLVX-shRNA1 at 16 ℃ overnight, wherein the connected reaction systems are shown in Table 4.

TABLE 4

(5) And (3) transformation: mu.L of each ligation product was transformed into 100. mu.L of JM107 competent cells: and uniformly mixing the ligation product and competent cells, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 70s, immediately placing on ice for 2min, adding 150 mu L of TB culture medium preheated to room temperature, carrying out shake culture at constant temperature of 37 ℃ for 1h at 250rpm, taking 100 mu L of TB culture medium by using a pipette, uniformly coating the TB culture medium on a 100 mu g/mL Ampicillin-resistant TB plate, inverting, and carrying out culture in a constant temperature incubator at 37 ℃ overnight.

(6) Positive clone plasmid extraction and enzyme digestion identification

A single clone on a TB plate is picked up, added into a TB culture medium for culturing, a recombinant plasmid is extracted by using a plasmid miniprep kit, and then the plasmids KpnI + BamHI and KpnI + EcoRI are subjected to enzyme digestion and identification, and the result is shown in figure 1.

In FIG. 1, the marker lane corresponds to DNA marker; lane K-ras-1(K + E) corresponds to the product of the ligation of pLVX-shRNA1 and K-ras-shRNA1 by transformation and then double digestion with EcoRI and KpnI; lane K-ras-1(K + B) corresponds to the product of the ligation of pLVX-shRNA1 and K-ras-shRNA1, transformed, then double digested with BamH1 and KpnI. The lane of K-ras-2(K + B) corresponds to a product obtained by transforming the ligation product of pLVX-shRNA1 and K-ras-shRNA2 and then carrying out double enzyme digestion on BamH1 and KpnI; lane K-ras-2(K + E) corresponds to the product of the ligation of pLVX-shRNA1 with K-ras-shRNA2, transformed, then EcoRI, KpnI double digested. The lane of K-ras-3(K + B) corresponds to a product obtained by transforming the ligation product of pLVX-shRNA1 and K-ras-shRNA3 and then carrying out double enzyme digestion on BamH1 and KpnI; lane K-ras-3(K + E) corresponds to the product of the ligation of pLVX-shRNA1 and K-ras-shRNA3 by transformation and then double digestion with EcoRI and KpnI; lane shRNAC (K + B) corresponds to the product of the ligation product of pLVX-shRNA1 and shRNAC after transformation and double digestion with BamH1 and KpnI; lane shRNAC (K + E) corresponds to the product of the ligation of pLVX-shRNA1 with shRNAC, transformed, and then EcoRI, KpnI double-digested.

The pLVX-shRNA1 has a BamH1 restriction site, a KpnI restriction site and an EcoRI restriction site, but after the EcoRI restriction site is combined with the cohesive end of EcoRI of the shRNA to form the shRNA recombinant vector, the EcoRI restriction site cannot be cut by EcoRI enzyme again. Therefore, when the shRNA recombinant vector is cut by EcoRI and KpnI, only one cutting site of KpnI is cut, and only one band appears during electrophoresis. Therefore, it was preliminarily determined from FIG. 1 that K-ras-shRNA1, K-ras-shRNA2, K-ras-shRNA3 and shRNAC were successfully inserted into pLVX-shRNA 1.

(7) Sequencing: and (3) selecting bacteria of the positive clones after enzyme digestion identification for culturing, and performing sequencing identification on the bacterial liquid after culture, wherein sequencing is performed by Jinweizhi company, and the sequencing identification result is shown in figures 2-5. FIG. 2 is a partial sequencing diagram of a recombinant vector (i.e., pLVX-K-ras-shRNA1) composed of K-ras-shRNA1 and pLVX-shRNA 1; FIG. 3 is a partial sequencing diagram of a recombinant vector (i.e., pLVX-K-ras-shRNA2) composed of K-ras-shRNA2 and pLVX-shRNA 1; FIG. 4 is a partial sequencing diagram of a recombinant vector (i.e., pLVX-K-ras-shRNA3) composed of K-ras-shRNA3 and pLVX-shRNA 1; FIG. 5 is a partial sequence chart of a recombinant vector comprising shRNAC and pLVX-shRNA1 (i.e., pLVX-shRNAC).

As shown in FIGS. 2 to 5, K-ras-shRNA1, K-ras-shRNA2, K-ras-shRNA3 and shRNAC were successfully inserted into pLVX-shRNA 1.

Example 2

Preparation of lentivirus for specifically inhibiting K-ras gene expression

(1) Packaging of lentivirus: according to Lenti-X^TMThe Lentiviral Expression Systems kit comprises the steps of respectively packaging lentiviruses with pLVX-K-ras-shRNA1, pLVX-K-ras-shRNA2, pLVX-K-ras-shRNA3 and pLVX-shRNAC to obtain corresponding K-ras-shRNA1 virus, K-ras-shRNA2 virus, K-ras shRNA-3 virus and K-ras-C virus. The specific operation is as follows:

a. at 37 deg.C, 5% CO₂293T cells were cultured in large numbers under the conditions and the digested cells were plated evenly.

b. When the fusion rate of the cells was 90%, transfection was prepared. Fully and uniformly mixing the transfection reagent, the packaging plasmid,

After the objective plasmid was obtained, the plasmid was dropped into a petri dish for transfection.

c. 293T cells were removed 48h after transfection and viral supernatants were collected.

(2) Concentrating and purifying the lentiviruses obtained in the step (1), wherein the concentration and purification of each lentivirus are as follows: centrifuging the virus at 10000RPM and 4 ℃ for 10min, and collecting the virus supernatant. Placing the virus supernatant in an ultracentrifuge centrifuge tube, balancing the centrifuge tube, centrifuging the virus at 50000RPM and 4 ℃ for 2h, removing the supernatant, retaining the precipitated virus, adding 1mL of PBS to dissolve the virus, and filtering the dissolved virus by using a 0.22 mu m filter. Finally, the filtered virus is tested for titer and packaged.

(3) Calculating the titer of each lentivirus obtained in the step (2), specifically:

a. cell preparation: 293 cells were cultured and seeded into 48-well plates with 105 cells per well at 37 ℃ with 5% CO₂The culture was continued for 18 h.

b. Preparation of Virus dilution solution 20. mu.L of the virus was diluted to 200. mu.L of DMEM medium, and the virus was sequentially diluted 10 × (10^-3、10^-4、10^-5、10^-6、10^-7) The diluted virus was mixed well and added to each of 48 wells at 100. mu.L/well, 37 ℃ with 5% CO₂The culture was continued for 48 h.

c. The results were observed and titers calculated: and (3) counting the number of the fluorescent cells in each well by using a fluorescence inverted microscope, finding out the count of the wells with two dilution concentrations with the minimum number of the fluorescent cells, and calculating the titer of the virus.

(4) And (3) detecting interference effect of lentivirus:

a. inoculation of HBE cells into six well plates, 3 × 10⁵Each cell is cultured for 18h to reach about 50% cell fusion degree, 10 microliter is added to obtain 10 titer⁸Viral supernatant TU/mL.

b. The cells were cultured for an additional 24h, then the lentiviral-containing medium was removed and fresh RPIM1640 complete medium was added for an additional 24 h.

c. RNA was extracted from each group of cells and reverse transcribed into cDNA.

d. The cDNA was used as a template to quantitatively detect the relative expression level of K-ras gene by fluorescence, and the fluorescence PCR system is shown in Table 5. The nucleotide sequence of the PCR Forward primer in Table 5 is 5'-GCGTAGGCAAGAGTGCCTTGA-3' (SEQ ID NO.17), and the nucleotide sequence of the PCR Reverse primer is 5'-GACCTGCTGTGTCGAGAATATCCA-3' (SEQ ID NO. 18). Reaction conditions of fluorescent PCR: melting experiments were carried out in the range of 55 ℃ to 95 ℃. 95 ℃ for 30s, 1 cycle, 55 ℃ for 30s, 40 cycles, 95 ℃ for 5s, 60 ℃ for 1min, 95 ℃ for 15 s.

TABLE 5

e. After the reaction is finished, a standard curve, a melting curve and a relative quantitative value are obtained. And analyzing the relative expression quantity of the K-ras gene of each group of cells according to the detected data. The results are shown in table 6 and fig. 6. Group "C" in FIG. 6 corresponds to K-ras-C virus; the "r 1" group corresponds to K-ras-shRNA1 virus; the "r 2" group corresponds to K-ras-shRNA2 virus; the "r 3" group corresponds to the K-ras-shRNA3 virus. The ordinate of FIG. 6 shows the relative expression level of K-ras gene.

TABLE 6

As can be seen from Table 6 and FIG. 6, the K-ras-shRNA1 virus, the K-ras-shRNA2 virus and the K-ras-shRNA3 virus can effectively and specifically inhibit the K-ras gene expression, wherein the K-ras-shRNA3 virus has the best interference effect.

Example 3

(1) Respectively constructing corresponding K-ras gene silent cell strains by using K-ras-shRNA3 virus and K-ras-C virus; the specific operation of each K-ras gene silencing cell strain is as follows:

a. HBE cell seeded in six well plates, 3 × 10⁵After culturing for 18h, the cell fusion degree reaches about 50% per well, and 10. mu.L (1 × 10) is added⁸TU/mL) of the corresponding virus supernatant.

c.48h later, HBE cells were infected with virus tagged with green fluorescent protein and the effect of virus infection was observed under a fluorescent inverted microscope. Then 1. mu.g/mL Puromycin (Puromycin) was added to the virus-infected cells. The liquid is changed every 2d, and the screening time is 7 d.

d. And after the screening is finished, removing the complete culture medium containing Puromycin (Puromycin) in the culture flask, and adding a normal complete culture medium to enable the cells to grow normally to obtain the K-ras gene silent cell strain.

(2) Identifying the K-ras gene silencing cell strain obtained in the step (1) by using real-time fluorescent quantitative PCR (qPCR):

a. and (2) inoculating the HBE cells and the K-ras gene silencing cells obtained in the step (1) into a 6-well plate respectively. The cell density reaches 80% >, EAt 90%, total RNA of each group of cells was extracted using RNeasy Mini Kit, mRNA was reverse-transcribed into cDNA using PrimeScrip rtreagen Kit, reverse transcription conditions: 15min at 37 ℃; 5s at 85 ℃; 4 ℃ and infinity. After completion of reverse transcription, 50. mu.L of RNase Free dH was added₂The cDNA was diluted O and stored at-20 ℃ for later use in assays.

b. Taking 1 mu L of cDNA of each group of cells as a template, taking GAPDH as an internal reference, and detecting the relative expression quantity of the K-ras gene by qPCR, wherein the primer sequence of the K-ras gene is as follows: 5'-GCGTAGGCAAGAGTGCCTTGA-3' (SEQ ID No.17) and 5'-GACCTGCTGTGTCGAGAATATCCA-3' (SEQ ID No. 18); the primer sequence of GAPDH is: 5'-TCTGACTTCAACAGCGACACC-3' (SEQ ID No.19) and 5'-CTGTTGCTGTAGCCAAATTCGT-3' (SEQ ID No. 20); setting reaction conditions: 30s at 95 ℃, 1 cycle, 30s at 55 ℃, 40 cycles at 95 ℃, 5s at 60 ℃, 1min at 95 ℃ and 15s at 95 ℃. SYBR Primescript RT-PCR Kit was used to detect the relative expression of K-ras gene in HBE cells and K-ras gene-silenced HBE cells, and the qPCR results are shown in FIG. 7. The ordinate of FIG. 7 shows the relative expression level of K-ras gene.

As can be seen from FIG. 7, the K-ras gene expression of HBE cell strain after K-ras gene silencing is obviously inhibited, and the gene expression level is reduced by 80.5% compared with HBE cell.

(3) Western blot identification of K-ras gene silencing cell strain

Respectively taking 1 bottle (25 cm) of HBE cells and K-ras gene silencing cells (corresponding to K-ras-shRNA3)²Cell culture flask), removing culture medium, washing with cold PBS 3 times, adding 500 μ L cell lysate, scraping the lysed cells from the flask wall quickly with a cell scraper, collecting protein, Loading into 500 μ L EP tube, further lysing at 4 deg.C for 30min, 12000rpm, centrifuging at 4 deg.C for 20min, finally adding 5 × SDS-PAGE Sample Loading Buffer, denaturing at 100 deg.C for 5min, performing 12% SDS-polyacrylamide gel electrophoresis, transferring the protein to PVDF membrane, sealing with 5% skimmed milk powder for 1h, adding K-ras antibody and GAPDH antibody respectively, incubating at 4 deg.C overnight at room temperature, washing the membrane with TBST for 3 times, adding secondary antibody once every 10min, incubating at room temperature for 1h, washing the membrane with TBST for 3 times, washing the membrane once every 10min, adding Western blot chemiluminescence reagent, and performing imaging analysis, wherein 1 indicates that the blot result is shown in FIGS. 8-9Untreated HBE cell group, 2 refers to HBE silent cell strain group constructed by K-ras-C virus, 3 refers to HBE silent cell strain group constructed by K-ras-shRNA3 virus, and the ordinate of FIG. 9 is relative content of K-ras protein.

As can be seen from FIGS. 8 to 9, the Western blot results are consistent with the Q-PCR results, and the K-ras protein expression of the HBE cell strain after the K-ras gene is silenced is reduced by 58.9%. The results show that the K-ras gene silencing cell strain is successfully constructed.

Example 4

Effect of specific inhibition of K-ras gene expression on apoptotic genes

HBE cells and K-ras gene silencing cells (HBE cell strains silenced by K-ras-shRNA3) are used as experimental objects, high-dose (50 mu g/mL) PM2.5 is used for poisoning for 24h (PM2.5 is fine particulate matters in haze and contains a plurality of health-harmful components such as heavy metal polycyclic aromatic hydrocarbon and the like, PM2.5 in example 4 is collected in Shanxi Taiyuan), and a blank control group is set at the same time; detecting the relative expression quantity of the oncogenes (c-myc, c-fos, k-ras and p53) and the apoptosis genes (Caspase-3 and Caspase-8) by fluorescence quantitative PCR, wherein the primer sequence of the c-myc of the Q-PCR is as follows: 5'-CCTGGTGCTCCATGAGGAGA-3' (SEQ ID No.21) and 5'-TCCAGCAGAAGGTGATCCAGAC-3' (SEQ ID No.21), the primer sequence for c-fos is: 5'-TCTTACTACCACTCACCCGCAGAC-3' (SEQ ID No.23) and 5'-GGAATGAAGTTGGCACTGGAGA-3' (SEQ ID No.24), the primer sequence of k-ras is: 5'-GCGTAGGCAAGAGTGCCTTGA-3' (SEQ ID No.18) and 5'-GACCTGCTGTGTCGAGAATATCCA-3' (SEQ ID No.18), the primer sequence of p53 is: 5'-AGAGCTGAATGAGGCCTTGGAA-3' (SEQ ID No.25) and 5'-GAGTCAGGCCCTTCTGTCTTGAAC-3' (SEQ ID No.26), the primer sequence of Caspase-3 is: 5'-GACTCTGGAATATCCCTGGACAACA-3' (SEQ ID No.27) and 5'-AGGTTTGCTGCATCGACATCTG-3' (SEQ ID No.28), the primer sequence of Caspase-8 is: 5'-CAAATGCAAACTGGATGATGAC-3' (SEQ ID No.29) and 5'-AGCAGGCTCTTGTTGATTTGG-3' (SEQ ID No. 30). The results of the fluorescent quantitative PCR are shown in FIGS. 10 to 15.

FIG. 10 is a graph showing the results of the effect of PM2.5 on K-ras gene expression; FIG. 11 is a graph showing the results of the effect of PM2.5 on c-fos gene expression; FIG. 12 is a graph of the results of the effect of PM2.5 on c-myc gene expression; FIG. 13 is a graph showing the results of the effect of PM2.5 on the expression of p53 gene; FIG. 14 is a graph showing the results of the effect of PM2.5 on Caspase-3 gene expression; FIG. 15 is a graph showing the results of the effect of PM2.5 on Caspase-8 gene expression; the ordinate in FIGS. 10 to 15 shows the relative expression amounts of K-ras, c-fos, c-myc, p53, Caspase-3 and Caspase-8 in that order. In fig. 10 to 15, a, c, and e represent p <0.05, and b, d, and f represent p < 0.01.

As can be seen from FIGS. 10 to 15, the expression levels of the target genes in K-ras gene-silenced cells (K-ras silent cells) were respectively reduced by 37.5%, 33.7% and 46.9% in the expression levels of the oncogenes c-myc, c-fos and K-ras in the PM 2.5-infected group, 11.6% in the expression level of the oncogene p53 and 28.8% and 37.7% in the expression levels of the pro-apoptotic genes Caspase-3 and Caspase-8, respectively, as compared with untreated normal HBE cells.

The results show that the K-ras gene-silenced HBE cells constructed in example 3 have significant changes in the expression of oncogenes and apoptotic genes after PM2.5 contamination treatment compared with untreated normal HBE cells, and the K-ras gene plays an important role in apoptosis and canceration of HBE cells caused by PM 2.5.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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Claims

1. An RNAi molecule specifically inhibiting the expression of the K-ras gene comprising a double-stranded RNA domain consisting of an antisense strand and a sense strand consisting of the nucleotide sequence represented by SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5 hybridized to said antisense strand, said antisense strand being hybridized to said sense strand under stringent hybridization conditions.

2. The RNAi molecule specifically inhibiting K-ras gene expression of claim 1, wherein the nucleotide sequence of the sense strand of the RNAi molecule is represented by SEQ ID No.1, and the nucleotide sequence of the antisense strand of the RNAi molecule is represented by SEQ ID No. 2;

3. The RNAi molecule specifically inhibiting K-ras gene expression according to claim 1 or 2, wherein the sense strand of the RNAi molecule is linked to the antisense strand of the RNAi molecule through a stem-loop structure.

4. A recombinant vector for specifically inhibiting K-ras gene expression is characterized by comprising a vector and a core fragment inserted in the vector, wherein the core fragment comprises a target sequence, a stem-loop structure sequence, a complementary sequence of the target sequence and a termination site which are sequentially connected, and the nucleotide sequence of the target sequence is shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5.

5. The recombinant vector for specifically inhibiting the expression of K-ras gene as claimed in claim 4, wherein the vector is a lentiviral vector; the nucleotide sequence of the sense strand of the core fragment is shown as SEQ ID No.7, and the nucleotide sequence of the antisense strand of the core fragment is shown as SEQ ID No. 8;

6. A construction method of a recombinant vector for specifically inhibiting K-ras gene expression is characterized by comprising the following steps:

providing a core fragment, wherein the core fragment comprises a target sequence, a stem-loop structure sequence, a complementary sequence of the target sequence and a termination site which are sequentially connected, and the nucleotide sequence of the target sequence is shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5; and

7. A recombinant engineering bacterium, which is characterized in that the recombinant engineering bacterium contains the RNAi molecule which specifically inhibits the expression of the K-ras gene and is disclosed in any one of claims 1-3;

or, the recombinant engineering bacteria contain the recombinant vector for specifically inhibiting the expression of the K-ras gene as claimed in any one of claims 4 to 5.

8. A lentivirus for specifically inhibiting K-ras gene expression is prepared by the following steps:

9. Use of the RNAi molecule specifically inhibiting K-ras gene expression as defined in any one of claims 1 to 3, the recombinant vector specifically inhibiting K-ras gene expression as defined in any one of claims 4 to 5, the recombinant engineered bacterium as defined in claim 7 or the lentivirus specifically inhibiting K-ras gene expression as defined in claim 8 for the preparation of a medicament for treating diseases associated with abnormal K-ras gene expression.

10. A pharmaceutical composition, which comprises the RNAi molecule for specifically inhibiting the expression of K-ras gene as claimed in any one of claims 1 to 3, the recombinant vector for specifically inhibiting the expression of K-ras gene as claimed in any one of claims 4 to 5, the recombinant engineered bacterium as claimed in claim 7 or the lentivirus for specifically inhibiting the expression of K-ras gene as claimed in claim 8.