CN110016477B

CN110016477B - SiRNA of schistosoma japonicum NAT13 gene and application thereof

Info

Publication number: CN110016477B
Application number: CN201910205094.9A
Authority: CN
Inventors: 洪炀; 唐亚兰; 林矫矫; 郭晴晴; 陈程; 何宇婷; 陈阳; 贾静; 傅志强; 朱传刚; 刘金明; 李�浩; 陆珂
Original assignee: Shanghai Veterinary Research Institute CAAS
Current assignee: Shanghai Veterinary Research Institute CAAS
Priority date: 2019-03-18
Filing date: 2019-03-18
Publication date: 2022-04-12
Anticipated expiration: 2039-03-18
Also published as: CN110016477A

Abstract

The invention discloses a siRNA of a schistosoma japonicum NAT13 gene, which is selected from one or any combination of more than two pairs of the following components: nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2; nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO. 4; nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO. 8. The invention also discloses the application of siRNA of the schistosoma japonicum NAT13 gene. The siRNA of the schistosoma japonicum NAT13 gene can obviously inhibit the transcription of SjNAT13 gene, can obtain 38.91% of egg reduction rate in vitro interference and 37.66% of egg reduction rate in vivo interference, and is suitable for preparing medicines influencing the oviposition of schistosoma japonicum.

Description

SiRNA of schistosoma japonicum NAT13 gene and application thereof

Technical Field

The invention relates to the technical field of molecular biology and biological medicine, in particular to siRNA of a schistosoma japonicum NAT13 (N-acetyltransferase 13) gene and application thereof.

Background

Schistosomiasis japonica is a parasitic disease which is widely distributed and seriously harmful to both human and animals and is caused by schistosoma japonicum infection. The infected areas are mainly in China, Indonesia, Philippines and the like. China has made a great effort in controlling the prevalence of schistosomiasis through a half century of unscrupulous effort, but the intermediate host oncomelania in the regions of rivers, lakes and continents and in the mountainous areas is difficult to eliminate, the infection source of livestock is difficult to be effectively controlled, and the phenomenon of repeated infection of schistosomiasis in human and livestock is serious, so the prospect of comprehensively controlling the spread of schistosomiasis is still unhappy, and the method can be a long-term, difficult and complex task. Praziquantel is used as the only drug for treating schistosomiasis in a large scale at present, and has the advantages of high efficiency, low toxicity, convenient use, low price and the like, and is widely applied to the clinical treatment and prevention and treatment of schistosomiasis, and no new drug for treating schistosomiasis appears for more than 20 years. However, in recent years, the resistance of schistosoma mansoni to praziquantel has been reported, and the emergence of drug-resistant strains has attracted people's uneasiness and high attention. Therefore, researchers are urgently required to find new measures for preventing and treating schistosomiasis and new drugs for treating schistosomiasis.

NAT13 is a possible catalytic component of the ARD1A-NARG1 complex, which shows the activity of alpha (N-terminal) acetyltransferases. Its alias names are Naa50, San, NAT5, NatE. Naa50/San is an N-terminal acetyltransferase required during mitosis, during chromosome division. Although this enzyme is highly conserved in higher eukaryotes, its function in mitosis is poorly understood, Naa50/San was first found to function when Drosophila's sister chromatid aggregates, and more recently it was found to act as a negative regulator of microtubule polymerization through internal acetylation of beta tubulin.

RNA interference (RNAi) is a technological approach to regulate gene expression at the transcriptional level, and the technology has been remarkably developed in the fields of functional genomics, gene therapy, microbiological research, drug development, and the like, and is widely used for studies related to post-transcriptional gene silencing, and the like. The technology is characterized in that exogenous double-stranded RNA with small molecular weight is introduced into host cells to ensure that endogenous mRNA is subjected to specific degradation, and the expression of a modified target gene is blocked after transcription so as to cause the loss of a corresponding functional phenotype. So-called small interfering RNAs (siRNAs) inhibit translation of a target protein by directing a cleavage enzyme in a RISC complex to bind to a target sequence in the target mRNA, thereby cleaving the mRNA molecule from the middle of the target sequence. siRNAs usually consist of more than 20 nucleotides and are capable of specific post-transcriptional gene silencing. SiRNA has been widely used in the fields of gene expression regulation mechanism research and the like.

Disclosure of Invention

Aiming at the defects in the prior art, the technical problem to be solved by the invention is to provide the siRNA of the schistosoma japonicum NAT13 gene and the application thereof, wherein the siRNA can obviously inhibit the transcription of the NAT13 gene and can be used for preparing a medicine for influencing the oviposition of schistosoma japonicum.

In order to realize the purpose, the invention provides the siRNA of the schistosoma japonicum NAT13 gene, which adopts the following technical scheme:

an siRNA of a schistosoma japonicum NAT13 gene, wherein the siRNA is selected from any one or the combination of any two or more of the following pairs:

nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;

nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO. 4;

nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO. 6;

the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO. 8.

Preferably, the siRNA is a nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO. 2.

The invention also provides a medicine for influencing the oviposition of schistosome, and the active ingredients of the medicine are as follows: siRNA for inhibiting the expression of schistosoma japonicum NAT13 gene by RNA interference.

The siRNA is a nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO. 2.

The invention also provides the application of the siRNA of the schistosoma japonicum NAT13 gene in the preparation of drugs influencing the oviposition of schistosoma japonicum.

The invention has the beneficial effects that:

the siRNA of the schistosoma japonicum NAT13 gene can be used for interfering the transcription of the schistosoma japonicum NAT13 gene and reducing the gene expression and the egg reduction of schistosoma japonicum, and an in vitro interference experiment shows that the siRNA can obtain 38.91 percent of egg reduction rate, and an in vivo RNA interference experiment of a mouse shows that the siRNA can obtain 37.66 percent of egg reduction rate, so that the siRNA is suitable for preparing a medicament for influencing the oviposition of schistosoma japonicum.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a graph showing the effect of real-time quantitative PCR analysis on in vitro interference in example 1 of the present invention.

Detailed Description

The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes and modifications may be made without departing from the principles of the invention and these changes and modifications are to be considered within the scope of the invention.

In the following examples, the experimental procedures without specifying the specific conditions were generally carried out according to conventional conditions, as described in molecular cloning, A laboratory Manual (J. SammBruk, D.W. Lassel, Huang Peyer, Wan Jia seal, Zhu Hou, et al, 3 rd edition, Beijing: scientific Press, 2002).

The reagents and biomaterials used below were all commercial products unless otherwise specified.

Example 1 in vitro RNA interference

1.1 design of siRNA molecules

Four pairs of siRNA molecules (see Table 1 below) were designed based on the sequence of the Schistosoma japonicum NAT13 gene (GeneBank: FN317573.1), NC siRNA molecules were used as unrelated controls, and all siRNAs were designed and synthesized by Shanghai Jima pharmaceutical technology, Inc.

TABLE 1 siRNA sequences

1.2 Collection of polypides

The abdominal patch is used to attack infected Kunming mouse, each infected with about 200 pieces of Schistosoma japonicum cercaria. Dissecting and killing after 21 days of infection, collecting insect bodies by adopting a heart perfusion method, quickly cleaning the insect bodies once by using warm high-sugar DMEM containing double antibiotics, then washing the insect bodies for three times by using an insect body complete culture medium in a super clean bench, putting the insect bodies into a 6-hole plate, adding 3ml of the insect body complete culture medium into each hole, and culturing the insect bodies in an incubator at 37 ℃ overnight.

1.3 in vitro interference

The method adopts an electrotransfer method to carry out interference and screen small RNA interference molecules, and comprises the following specific steps:

(1) the siRNA was removed and then centrifuged at 12000g for 5min and diluted on ice (1OD + 125. mu.l DEPC H)₂O40 μ M), i.e. 125 μ L of DEPC H₂O resuspend 1OD siRNA, dissolve 40 u M sample, take to the cell room and prepare for the electric transfer. The power supply of the electrotransfer instrument is turned on, and the voltage and the electrotransfer time (LV: 125; pulses: 0.1; Interval: 0.1; polarity: unipular) are adjusted. Adding the preheated culture medium into a small round plate, transferring the worms from a 6-well plate into the small round plate with the culture medium, picking 5 pairs of worms into an electric rotating cup, removing residual culture medium, adding 70 mul of culture medium into the electric rotating cup, adding 30 mul of siRNA, uniformly mixing, and then carrying out electric rotation on an electric rotating instrument, wherein the final concentration of the electric rotation is 12 mul. The electroporated worms were placed in a new 6-well plate with pre-heated complete medium and cultured in a 37 ℃ incubator.

(2) The experiment is divided into 6 groups, namely Sj-514, Sj-48, Sj-154, Sj-575 interference groups, unrelated interference NC control groups and blank control groups. Three wells were repeated for each set.

(3) After the interference lasts for 72 hours, collecting polypide, extracting total RNA, carrying out reverse transcription to obtain cDNA, and detecting the change of the transcription level of the gene by using real-time quantitative PCR to judge the interference effect.

1.4 results

In vitro interfering siRNA interfering group, irrelevant interfering group, blank control group worm, RNA extraction, real-time quantitative PCR detection SjNAT13 gene interference situation, the results are shown in figure 1. As can be seen from the results in FIG. 1, Sj-514, Sj-48, Sj-154, Sj-575 interference groups all showed inhibition of SjNAT13 gene transcription compared with irrelevant interference groups and blank control groups; the siRNA with the number Sj-514 has the best interference effect, the interference effects of Sj-48 and Sj-154 are centered, the siRNA with the number Sj-575 has the second interference effect, the transcription of SjNAT13 gene is obviously inhibited, and then the siRNA with the number Sj-514 is selected for in vivo interference test.

In combination with the following table, in vitro interference experiments showed that the siRNA molecule interference group numbered Sj-514 achieved 38.91% egg reduction (P <0.05) compared to the blank control group; the unrelated group interfered with only 9.49% of the egg drop rate (P > 0.05).

TABLE 2 in vitro interference-induced egg reduction Effect

Group of	Egg number/insect (mean + -SD)	Rate of ova reduction
			Blank control	16283.33333±2332.369282	---
Extraneous interference	14737.5±2088.044673	9.49％
			Sj-514 interference group	9947.916667±1113.939975	38.91％

Example 2 in vivo RNA interference of accession number Sj-514

2.1 method step

15 six-week-old BALB/c mice were divided into three groups, namely a blank control group, an irrelevant interference control group and an siRNA interference group. The schistosoma japonicum cercaria was infected with the abdominal patch, and about 40 were infected per mouse. Starting on day 18 after infection, mice in the blank control group were injected into tail vein with 0.1ml of PBS solution (pH 7.4) every six days, mice in the irrelevant interference control group were injected into tail vein with 0.1ml of NC (1OD)/PBS (pH 7.4) solution every six days, and mice in the siRNA interference group were injected into tail vein with 0.1ml of SjNAT13 gene siRNA Sj-514(1OD)/PBS (pH 7.4) solution every six days. After 4 times of injection, the animals are killed by dissection at 42 days, washed by a hepatic portal vein infusion method, observed and counted, and the number of the animals and the number of eggs in the liver are counted.

2.2 results

The results of in vivo interference experiments show that after the siRNA with the code of Sj-514 is injected into the tail vein of the mouse, partial egg reduction rate is induced, and the results are shown in the table 3. Compared with a blank control group, the siRNA molecule with the number of Sj-514 interference group obtains the egg reduction rate of 37.66% (P <0.05), and the irrelevant interference group only has the egg reduction rate of 0.80% (P > 0.05).

TABLE 3 in vivo interference-induced ova-decreasing Effect

Group of	Egg number/insect (mean + -SD)	Rate of ova reduction
			Blank control	12768.45238±2727.104105	---
Extraneous interference	12666.12554±1609.434448	0.80％
			Sj-514 interference group	7959.989177±1172.57902	37.66％

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present 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.

Sequence listing

<110> Shanghai veterinary research institute of Chinese academy of agricultural sciences

<120> siRNA of Japanese blood fluke NAT13 gene and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (20)..(21)

<223> t

<400> 1

gcgcaguugc uuucuacaat t 21

<210> 2

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (20)..(21)

<223> t

<400> 2

uuguagaaag caacugcgct t 21

<210> 3

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (20)..(21)

<223> t

<400> 3

gcuguacgua ccgaacaaat t 21

<210> 4

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (20)..(21)

<223> t

<400> 4

uuuguucggu acguacagct t 21

<210> 5

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (20)..(21)

<223> t

<400> 5

ucaagcaguu uagacugaut t 21

<210> 6

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (20)..(21)

<223> t

<400> 6

aucagucuaa acugcuugat t 21

<210> 7

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (20)..(21)

<223> t

<400> 7

ccgacgcauu cacccucaat t 21

<210> 8

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (20)..(21)

<223> t

<400> 8

uugaggguga augcgucggt t 21

<210> 9

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (20)..(21)

<223> t

<400> 9

uucuccgaac gugucacgut t 21

<210> 10

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (20)..(21)

<223> t

<400> 10

acgugacacg uucggagaat t 21

Claims

1. The siRNA of the schistosoma japonicum NAT13 gene is characterized in that the siRNA is selected from any one or the combination of any two or more than two pairs of the following:

nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;

nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO. 4;

nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO. 6;

the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO. 8.

2. The siRNA of the NAT13 gene of Schistosoma japonicum according to claim 1, wherein the siRNA comprises the following components: the siRNA is a nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO. 2.

3. A drug for influencing the oviposition of Schistosoma japonicum containing NAT13 gene is characterized in that the active ingredients of the drug are as follows: siRNA for inhibiting the expression of schistosoma japonicum NAT13 gene through RNA interference; the siRNA is a nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO. 2.

4. The use of siRNA of the NAT13 gene of Schistosoma japonicum according to claim 2 in the preparation of a medicament for reducing oviposition of Schistosoma japonicum containing NAT13 gene.

5. An application of siRNA of schistosoma japonicum NAT13 gene in preparing medicine for influencing the oviposition of schistosoma japonicum containing NAT13 gene; the siRNA is a nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO. 2.