Preparation method of anti-aspergillus niger siRNA and complex preparation thereof

Abstract

The application relates to the technical field of biological medicine, in particular to a preparation method of anti-aspergillus niger siRNA and a complex preparation thereof, wherein the anti-aspergillus niger siRNA comprises siR266 for a PmrA gene and / or siR505 for a SarA gene, the sequence of a sense strand and an antisense strand of the siR266 is shown in SEQ ID NO: 3-4 respectively, and the sequence of a sense strand and an antisense strand of the siR505 is shown in SEQ ID NO: 7-8 respectively. Wherein, the complex preparation adopts protamine sulfate as a carrier, and forms a complex with the anti-aspergillus niger siRNA. The complex preparation prepared by the application can effectively inhibit aspergillus niger, and can be applied to food preservation, postharvest protection of fruits and vegetables, and mildew prevention of daily necessities and the like, and is suitable for developing corresponding anti-fungal products.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine technology, and in particular to a preparation method of anti-Aspergillus niger siRNA and a complex preparation thereof. BACKGROUND

[0002] Aspergillus niger belongs to Aspergillus, is a common saprophytic filamentous fungus in the order of Chaetomycetales and family of Chaetomiaceae. It is not only an important production strain in various industrial fermentation processes, but also a common food spoilage microorganism and opportunistic human pathogen. The fungus is widely distributed in nature, commonly found in soil, agricultural products and food, and can cause serious postharvest diseases (such as black mold disease), significantly reduce the storage quality of fruits and vegetables and cause significant economic losses. In addition, A. niger has a certain pathogenic potential to insects, animals and humans, and can cause various infectious diseases. At present, the main means to control fungal contamination still relies on chemical preservatives (such as benzoic acid, sorbic acid, propionic acid and their salts) and antifungal drugs (such as azoles, echinocandins and polyenes, etc.). However, the widespread use of chemical fungicides poses potential risks to food safety, ecological environment and human health; at the same time, the long-term application of antifungal drugs also easily leads to the increase of fungal drug resistance, and the prevention and control effect gradually decreases. Therefore, developing efficient, safe and not easily inducing drug resistance green antifungal strategies has become an urgent need in the prevention and control of postharvest diseases of agricultural products and public health management.

[0003] RNA interference (RNAi) is a highly conserved gene silencing mechanism in eukaryotes, which can mediate the specific degradation or translation inhibition of target mRNA through small interfering RNA (siRNA), so as to realize the regulation of gene expression. The siRNA-based antifungal strategy has the advantages of flexible sequence design, strong action specificity and environmental friendliness, and shows broad application prospects in the fields of medical nucleic acid drugs, agriculture and food preservation. However, naked siRNA faces many challenges in practical application: it is easy to be degraded by nucleases, it is difficult to penetrate the cell membrane due to carrying strong negative charge, and the complex structure of fungal cell wall also seriously hinders its intracellular delivery, resulting in low gene silencing efficiency. Therefore, efficient delivery of siRNA is the key to improving its antifungal effect.

[0004] Protamine sulfate (PS) is a cationic polypeptide rich in arginine. Its high positive charge enables it to spontaneously bind to negatively charged siRNA through electrostatic interactions, forming a stable complex. This complex can effectively neutralize the charge of siRNA and protect it from nuclease degradation, thereby improving the stability and delivery efficiency of siRNA. Protamine sulfate has good biological safety and has been approved by the FDA for use in the medical field. Currently, protamine sulfate is mainly used as a nucleic acid carrier in gene therapy and tumor vaccine fields, but its application in fungal control, especially in the construction of siRNA complexes to inhibit Aspergillus niger, has not been reported. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of anti-Aspergillus niger siRNA and its complex preparation. The complex preparation prepared by the present application is an anti-Aspergillus niger inhibitor based on protamine sulfate / siRNA complex, which can significantly inhibit the growth of Aspergillus niger, thereby providing a green and safe new control strategy.

[0006] To achieve the above purpose, the present application provides the following technical solutions:

[0007] The siRNA designed by the present application takes the key genes (pmrA, sarA) of Aspergillus niger as the target.

[0008] Based on the sequence of the target mRNA, the present application uses two different principle design tools, DSIR of the first generation algorithm and siDirect of the second generation algorithm, to screen siRNA sequences. The designed candidate sequences need to meet the following conditions at the same time: ranked in the top five in the DSIR tool, and meet the classic design principles of Ui-Tei, Reynolds and Amarzguioui in the siDirect tool. The obtained candidate siRNA sequences include sense strand (SS Sequence, also known as passenger strand) and antisense strand (AS Sequence, also known as guide strand).

[0009] The candidate siRNA for pmrA gene is siR267, siR266 and siR117. The siR267 is composed of a sense strand with a nucleotide sequence as shown in SEQ ID NO. 1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 2. The siR266 is composed of a sense strand with a nucleotide sequence as shown in SEQ ID NO. 3 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 4. The siR117 is composed of a sense strand with a nucleotide sequence as shown in SEQ ID NO. 5 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 6.

[0010] The candidate siRNAs for the sarA gene are siR505 and siR507 respectively. The siR505 is composed of a sense strand with a nucleotide sequence as shown in SEQ ID NO. 7 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 8. The siR507 is composed of a sense strand with a nucleotide sequence as shown in SEQ ID NO. 9 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 10.

[0011] In order to overcome the problem that the naked siRNA is difficult to penetrate the cell membrane and cell wall of fungi and is easily degraded by nucleases due to the negative charge, the application provides a complex preparation prepared by using the anti-aspergillus niger siRNA, specifically, using protamine sulfate as a carrier to form a complex with the siRNA to effectively protect and deliver the siRNA.

[0012] Further, the siR266 and the siR505 are mixed with the protamine sulfate in equal volume to form protamine sulfate-siRNA complexes, which are named as PS-siRNA2 and PS-siRNA3 respectively.

[0013] Further, the siR266 and the siR505 are mixed with the protamine sulfate in equal volume to form protamine sulfate-siRNA complexes, which are named as PS-siRNA2 and PS-siRNA3 respectively.

[0014] Further, the temperature of the incubation is room temperature.

[0015] Further, the incubation time is 30 min.

[0016] The complex preparation of the application can be used for preparing a medicine for inhibiting aspergillus niger. The complex preparation can effectively inhibit aspergillus niger and can be applied to food preservation, postharvest protection of fruits and vegetables, and mold prevention of daily necessities, and is suitable for developing corresponding antifungal products.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] (1) The complex preparation of the application shows excellent inhibitory and killing effects on aspergillus niger. The minimum inhibitory concentration (MIC) of the PS-siRNA2 complex is 0.625 μM, and the minimum fungicidal concentration (MFC) is 1.25 μM; and the MIC and MFC values of the PS-siRNA3 complex are both 0.625 μM.

[0019] (2) The application based on the RNAi gene silencing mechanism acts on specific target mRNA and is not easy to cause fungal drug resistance, and has good sustainable application potential.

[0020] (3) The protamine sulfate is an FDA-approved pharmaceutical excipient, and the siRNA itself is biocompatible, so the compound thereof has no toxic side effects and is a green and safe preparation.

[0021] (4) The preparation process of the compound preparation is simple in operation, mild in conditions and free of complex equipment. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The loop structure type and the prediction probability distribution of the target region of the candidate siRNA (siR267, siR266 and siR117) designed for the pmrA gene are shown, wherein [A], [B] and [C] respectively represent the prediction results of the hairpin loop, the convex loop and the inner loop.

[0023] Figure 2 The loop structure type and the prediction probability distribution of the target region of the candidate siRNA (siR505 and siR507) designed for the sarA gene are shown, wherein [A], [B] and [C] respectively represent the prediction results of the hairpin loop, the convex loop and the inner loop.

[0024] Figure 3 The antibacterial effect of each treatment group on Aspergillus niger at different siRNA final concentrations is shown. The treatment groups include: CK (blank control), CY (protamine sulfate only), PS-siRNA2 and PS-siRNA3 (protamine sulfate / siRNA compound). [A], [B] and [C] respectively correspond to the experimental results of siRNA concentrations of 2.5 μM, 5 μM and 10 μM.

[0025] Figure 4 The comparison results of the antibacterial circle diameters of each treatment group on Aspergillus niger at different siRNA final concentrations are shown. The treatment groups include: CK (blank control), CY (protamine sulfate only), PS-siRNA2 and PS-siRNA3 (protamine sulfate / siRNA compound). Wherein, a, b, c and d represent the significance difference conditions, and different letters represent significant difference, and the same letter represents no significant difference.

[0026] Figure 5 The minimum inhibitory concentration (MIC) determination results of the PS-siRNA2 compound on Aspergillus niger are shown.

[0027] Figure 6 The minimum inhibitory concentration (MIC) determination results of the PS-siRNA3 compound on Aspergillus niger are shown.

[0028] Figure 7 The minimum fungicidal concentration (MFC) determination results of the PS-siRNA2 compound on Aspergillus niger are shown.

[0029] Figure 8 The minimum fungicidal concentration (MFC) of the PS-siRNA3 complex against A. niger was determined. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work are within the protection scope of the present application.

[0031] Example 1 Design and screening of siRNA

[0032] The present application takes the key genes pmrA and sarA of A. niger as the targets of siRNA design to achieve the purpose of inhibiting the growth of the bacterial body. The reference sequences of the target genes pmrA and sarA are derived from GenBank accession numbers XM_059746806.1 and XM_001388795.3, respectively. Based on the above sequences, two algorithm tools, DSIR and siDirect, are used for rational design of siRNA. The preliminary screening conditions are as follows: the candidate sequence needs to be in the top five in the DSIR score and at the same time meet the Ui-Tei, Reynolds and Amarzguioui criteria in the siDirect tool. The final candidate siRNA sequences that meet all the design requirements are listed in Table 1 below.

[0033] Table 1 Candidate siRNA designed for target genes and sequences thereof

[0034]

[0035] Based on the foregoing screening criteria, the candidate siRNA sequences are subjected to multi-dimensional screening, and the specific steps are as follows: (1) using the GC content calculator to analyze the GC content of each siRNA sequence to ensure that it is within the range of 30%-60%; (2) predicting and calculating the folding free energy of the guide strand through the RNAstructure web server; (3) calculating the binding free energy of siRNA and target mRNA using the RNAstructure web server; (4) analyzing the possible circular structure of the candidate siRNA corresponding to the target sequence using the Sfold Web server, and preferentially selecting the binding site in the relatively stable structure region, and the specific circular structure probability distribution is shown in Figure 1 and Figure 2(5) using siRNAPred tool to score the effectiveness of the candidate sequence, and prefer to score higher. After the above screening and evaluation, siR266 (named siRNA2) targeting pmrA gene and siR505 (named siRNA3) targeting sarA gene were selected as the sequences used in subsequent experiments, and the related detailed screening data are shown in Table 2.

[0036] Table 2. Screening data table of candidate siRNA

[0037]

[0038] Example 2. Preparation of protamine sulfate / siRNA complex

[0039] The siRNA2 and siRNA3 sequences screened and determined were synthesized and purified by GenScript Biotech (Shanghai) Co., Ltd. Protamine sulfate was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0040] The hepes buffer was used to prepare the stock solution respectively: the siRNA stock solution concentration was 20 μM, and the protamine sulfate stock solution concentration was 5 mg / mL. According to the nitrogen-phosphorus ratio (N / P = 20:1), the corresponding volume of protamine sulfate solution and siRNA solution were mixed in equal volume, incubated at room temperature for 30 minutes to form protamine sulfate / siRNA complex (denoted as PS-siRNA2 and PS-siRNA3 respectively).

[0041] The complex concentration was expressed by the final concentration of siRNA. The complex solution with a final concentration of siRNA of 2.5 μM, 5 μM and 10 μM was prepared respectively, and the corresponding final concentration of protamine sulfate was 105 μM, 210 μM and 420 μM respectively. That is, three concentration gradients were set for each complex (PS-siRNA2 and PS-siRNA3).

[0042] Example 3. Inhibition effect of protamine sulfate / siRNA complex on Aspergillus niger

[0043] The Aspergillus niger strain stored at -80℃ was inoculated in PDA medium and cultured at 28℃ for 7 days until the colony was mature and produced a large number of spores. The spores were eluted with sterile normal saline, and the spore suspension was obtained after filtering with sterile absorbent cotton. The spore concentration was adjusted to 1×10 7 CFU / mL using a hemocytometer.

[0044] The punching method was used to determine the antibacterial effect. 100 μL of spore suspension with a concentration of 1×10 6The spore suspension of 1 x 10 7 CFU / mL was uniformly coated on the surface of PDA plate with a sterile coating rod, and after punching with a sterile 6 mm puncher, 50 μL of the sample to be tested was added to each hole. The experiment was divided into 4 groups: CK group (blank control, buffer was added), CY group (containing only protamine sulfate), PS-siRNA2 group (complex prepared in Example 2) and PS-siRNA3 group (complex prepared in Example 2). Each group was tested according to the method described in Example 2 at siRNA final concentrations of 2.5 μM, 5 μM and 10 μM. The bacteriostatic effect was observed at 48 h and 72 h, respectively.

[0045] The results showed that the CK group did not produce inhibition zone (the diameter of the inhibition zone was the hole diameter of 6 mm) at each concentration. With the increase of concentration, the bacteriostatic effect of CY group and two complex groups (PS-siRNA2, PS-siRNA3) was significantly enhanced. At all concentration conditions, the bacteriostatic effect of the complex group was significantly better than that of CY group and CK group (p<0.05), indicating that the introduction of siRNA can significantly enhance the inhibitory ability of protamine sulfate on Aspergillus niger, and the selected siRNA sequence has good inhibitory effect on Aspergillus niger. The bacteriostatic results are shown in Figure 3 , wherein [A], [B] and [C] correspond to the bacteriostatic conditions at 2.5 μM, 5 μM and 10 μM siRNA final concentration for 48 h and 72 h, respectively. The results of measuring the diameters of the inhibition zones of each group after 72 h of culture are shown in Figure 4 .

[0046] Example 4 Determination of the minimum bacteriostatic concentration of protamine sulfate / siRNA complex

[0047] The minimum bacteriostatic concentrations (MIC) of complexes PS-siRNA2 and PS-siRNA3 on Aspergillus niger were determined by 96-well plate microdilution method. The spore suspension of Aspergillus niger (initial concentration 1 x 10 7 CFU / mL) was diluted to 1 x 10 5CFU / mL as inoculum. The complex solution was serially diluted with buffer at the following concentration gradient: 2.5 μM, 1.25 μM, 0.625 μM, 0.313 μM, 0.156 μM, 0.078 μM, 0.039 μM, 0.020 μM, 0.010 μM, 0.005 μM. 100 μL of the diluted complex solution was added to each well, followed by 100 μL of the above-mentioned spore inoculum, so that the final volume in each well was 200 μL. The 11th well was a positive control (PC group, containing the spore inoculum and the culture medium, but not the complex), and the 12th well was a negative control (NC group, containing only the culture medium and the buffer). After the 96-well plate was incubated at 28°C for 72 hours, it was observed by naked eye. The lowest complex concentration at which no growth of A. niger was observed was determined as the MIC. The experimental results showed that both PS-siRNA2 and PS-siRNA3 exhibited significant inhibitory activity against A. niger, and the minimum inhibitory concentration (MIC) of each was 0.625 μM. Figure 5 and Figure 6 ).

[0048] Example 5 Determination of the Minimum Fungicidal Concentration of Protamine / SiRNA Complexes

[0049] The wells in which no growth of A. niger was observed in Example 4 (corresponding to a complex concentration of 2.5 μM, 1.25 μM, 0.625 μM, i.e. 4xMIC, 2xMIC and lxMIC groups) and the positive control group (PC) samples were each transferred to fresh PDA culture medium containing no complex, and incubated at 28°C for another 72 hours. The lowest complex concentration at which no growth of A. niger was observed was determined as the minimum fungicidal concentration (MFC). The results showed that the MFC of PS-siRNA2 complex was 1.25 μM (2xMIC), and the MFC of PS-siRNA3 complex was the same as the MIC, both being 0.625 μM. The experimental results are shown in Table 2. Figure 7 and Figure 8 .

[0050] Although the embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. An anti-Aspergillus niger siRNA, characterized in that: This includes siR266 targeting the PmrA gene and / or siR505 targeting the SarA gene. The sense and antisense sequences of siR266 are shown in SEQ ID NO:3-4, and the sense and antisense sequences of siR505 are shown in SEQ ID NO:7-8, respectively.

2. The compound preparation made using the anti-Aspergillus niger siRNA according to claim 1, characterized in that: Protamine sulfate was used as a carrier to form a complex with anti-Aspergillus niger siRNA.

3. The method for preparing the compound formulation according to claim 2, characterized in that: siR266 and siR505 were mixed with protamine sulfate in equal volumes and incubated to form protamine sulfate-siRNA complexes.

4. The method for preparing the compound formulation according to claim 3, characterized in that: SiR266 and SiR505 were mixed with protamine sulfate in equal volumes at a nitrogen-to-phosphorus ratio of 20:

1.

5. The method for preparing the compound formulation according to claim 4, characterized in that: The incubation temperature is room temperature.

6. The method for preparing the compound formulation according to claim 5, characterized in that: The incubation time is 30 minutes.

7. The use of the compound formulation of claim 2 in the preparation of a drug for inhibiting Aspergillus niger.

8. The application of the compound preparation according to claim 2 in food preservation, post-harvest protection of fruits and vegetables, and anti-mildew treatment of daily necessities.

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