TW202010410A - A microbial agent for controlling transmission of mosquito-borne diseases - Google Patents

Abstract

The present invention provides a polypeptide which comprises an amino acid sequence with trypsin like protease cutting site for digesting in larval gut, and short peptides of N- and C- terminus for stabling the amino acid sequence, in which, the amino acid sequence comprises at least one lysine (K) cutting site or arginine (R). The present invention is also related to a microbial agent for controlling transmission of mosquito borne diseases, which expresses a mosquito killing protein on cell membrane of bacterial carrier by using the polypeptide of present invention, comprising a bacterial carrier, the peptide, and a mosquitocidal toxin.

Description

Microbial preparation for controlling the spread of mosquito-borne diseases

The invention relates to a microbial preparation, in particular to a microbial preparation for controlling the spread of mosquito-borne diseases.

Taiwan, China, Southeast Asian countries, or other countries located in tropical and subtropical climate regions are prone to mosquito-borne diseases such as Japanese encephalitis, Zika virus, or dengue fever due to mosquitoes. Taking dengue fever as an example, China has listed dengue fever as the second type of infectious disease in the Republic of China in 1993. In the past 10 years, there have been 800 to 10,000 confirmed cases of dengue fever each year in our country. In 2015, dengue fever The number of confirmed cases has risen to more than 40,000. Although the current medical development and the proper implementation of policies, dengue fever cases are gradually decreasing, but with the impact of global warming, the areas where mosquitoes can multiply are gradually spreading. According to the statistics published by the Control Department, the number of confirmed cases of dengue fever in the Greater Taipei area has begun to be higher than that of Tainan and Kaohsiung since 2017.

Most of the mosquito repellent methods for the environment use large-scale spraying. One is that the insecticide is a chemical product, which has a great impact on the environment; both, the insecticide grows well in the water, and spraying the insecticide indirectly pollutes the water quality. The quality of domestic water and health impacts are worrying, and developing environmentally friendly mosquito killing methods is a top priority. At present, the whole world is committed to biological control work, advocating the use of the biological food chain and the relative relationship between natural enemies to control pests. The present invention is based on the concept of the food chain to design a kind of food that can be eaten by the mosquitoes. And to achieve the effect of killing mosquitoes.

Past studies have pointed out that Bacillus thuringiensis and Bacillus sphaericus have their own mosquito-killing proteins that can kill mosquitoes. There are also some insecticides made of Bacillus thuringiensis, but their The disadvantages are: 1. Bacillus thuringiensis is not an environmental probiotic, 2. Bacillus thuringiensis has mosquito killing characteristics only in the spore stage, 3. Bacillus thuringiensis preparation itself does not have the ability to replicate, therefore, the present invention provides a new concept Utilizing a polypeptide chain that can be digested by the enzymes of the intestine to express the mosquito toxin protein on the cell membrane binding protein of the cell membrane of environmental probiotics. The use of the bacterial carrier has the characteristics of self-proliferation in the environment, so that the insecticide has Reproducible, and the mosquito toxin protein can be released faster and more efficiently.

The "short peptide" in the present invention refers to a short peptide chain of 3 to 9 amino acid sequences.

The spirit of the present invention is to provide a platform for microbial preparations and express a mosquito toxin protein on a bacterial carrier, wherein the bacterial carrier and the mosquito toxin protein have different choices according to their purposes.

The object of the present invention is to provide an environmentally friendly novel microbial preparation for controlling the transmission of mosquito-borne diseases. The main concept is to use an environmental microorganism as a carrier and express a mosquito-killing protein on its cell membrane.

One of the main technical features of the present invention is a polypeptide chain, which includes an amino acid sequence with trypsin like protease cleavage for digestion of mosquito intestines, and short peptides at both ends for stabilization The amino acid sequence. Wherein, the amino acid sequence includes at least monoamine acid (K) or monoarginine (R).

Another main technical feature of the present invention is a microbial preparation that uses the polypeptide chain to connect the mosquito toxin to the cell membrane of a bacterial carrier to control the transmission of mosquito-borne diseases, including a bacterial carrier, a polypeptide chain, and a mosquito toxin Protein, wherein the mosquito poisoning protein refers to mosquito killing protein.

Microbial preparations according to the present invention, in a preferred embodiment, the carrier system is a bacterial probiotic environment, including the genus Bacillus (Bacillus), and photosynthetic bacteria and nitrifying bacteria.

According to the microbial preparation of the present invention, in a preferred embodiment, the amino acid sequence of the polypeptide chain includes at least an amine acid (K) or an arginine (R).

According to the microbial preparation of the present invention, in a preferred embodiment, the mosquito toxin protein includes first type mosquito killing protein (Mosquitocidal Toxin 1), second type mosquito killing protein (Mosquitocidal Toxin 2), third type mosquito killing protein Protein (Mosquitocidal Toxin 3), Cry protein (Cry family proteins), Cyt protein (Cyt family proteins), CLP protein (Cyclic lipopeptide protein), or any combination thereof.

According to the microbial preparation of the present invention, in a more preferred embodiment, the mosquito toxin protein is Mosquitocidal Toxin 2 (Mosquitocidal Toxin 2). Protein (Truncated Mosquitocidal Toxin 2), the effect is better.

According to the microbial preparation of the present invention, in a preferred embodiment, the polypeptide chain is attached to the cell membrane binding protein on the cell membrane of the bacterial carrier, wherein the cell membrane binding protein has different options depending on the bacterial carrier For example, in E. coli , the membrane binding protein can be AIDA protein or OmpA protein; if it is on B. subtilis , the membrane binding protein can be YhcR protein.

The main technical feature of the present invention further includes a recombinant gene, including a gene for a membrane binding protein that can be transcribed out of the microbial preparation, a polypeptide chain gene, and a mosquito poisoning protein gene, wherein the polypeptide chain gene is set in Between the cell membrane binding protein gene and the mosquito toxin protein gene.

According to the recombinant gene of the present invention, in a preferred embodiment, the polypeptide chain gene has a gene of amino acid sequence cleaved by trypsin like protease, wherein the gene of the amino acid sequence includes at least one Genes of lysine (K) or arginine (R).

According to the recombinant gene of the present invention, in a preferred embodiment, the mosquito toxin-killing gene line includes the first type mosquito-killing protein (Mosquitocidal Toxin 1) gene, the second type mosquito-killing protein (Mosquitocidal Toxin 2) gene, the first Mosquitocidal Toxin 3 gene, Cry family protein gene, Cyt family protein gene, CLP protein (Cyclic lipopeptide protein) gene, or any combination thereof.

According to the microbial preparation of the present invention, in a more preferred embodiment, the mosquito toxin protein is a Mosquitocidal Toxin 2 gene (Mosquitocidal Toxin 2) gene, wherein, when the mosquito toxin protein is truncated and modified, the second type kills Mosquito protein (truncated Mosquitocidal Toxin 2) gene, its effect is better.

According to the microbial preparation of the present invention, in a preferred embodiment, the membrane-binding protein gene has different choices depending on the bacterial carrier. For example, in E. coli , the membrane-binding protein gene may be Choose AIDA protein gene or OmpA protein gene; if it is on B. subtilis , then the membrane binding protein can choose YhcR protein gene.

The use of the microbial preparation of the present invention in the control of mosquito-borne infections is to use an appropriate amount of the present invention in water. Since the mosquito larvae-growth grows in water, especially the non-flowing water, the appropriate amount of the present invention is used. When the microbial preparation is cast into water, the microbial preparation of the present invention will be ingested while eating, and after the polypeptide chain is decomposed by its intestinal enzymes, the mosquito poisoning protein will be released accordingly, so as to achieve mosquito control to control mosquito The purpose of vector-borne diseases.

10‧‧‧Microbiological preparation for controlling the transmission of mosquito-borne diseases

101‧‧‧ Bacteria carrier

102‧‧‧ Membrane binding protein

103‧‧‧ polypeptide chain

104‧‧‧ Mosquito poison protein

20‧‧‧

201‧‧‧The intestinal enzymes

FIG. 1 is a diagram of an embodiment of the present invention.

Figure 2 is the recombinant genome of the fusion mosquito toxin protein gene used in the E. coli expression system test of the present invention. The KM4 recombinant gene is the recombinant gene experiment group 1 of the present invention; the KM6 recombinant gene is a polypeptide chain-free gene ( Experimental group 2 replaced by GGGSG); KM3 recombinant gene is a control group without polypeptide chain gene and membrane protein gene.

Figure 3 shows the results of the recombinant protein fusion of mosquito toxins used in the E. coli expression system test of the present invention. The left picture shows the results of SDS-PAGE, and the right picture shows the use of mouse-derived primary antibodies against GST tag. The result of Western blotting.

Fig. 4 shows the results of the poisoning effect of the present invention on the E. coli expression system for the Egyptian zebra larvae. Fig. A shows the implementation of KM4 and KM6 entering the larvae. Fig. B shows the results of the killing test of KM4 and KM6. .

FIG. 5 is a recombinant gene of the Bacillus subtilis expression system fused with mosquito-killing protein for B. subtilis performance test of the present invention.

FIG. 6 shows the results of the recombinant protein expression of the Bacillus subtilis expression system fused with the mosquito-killing protein used in the B. subtilis performance test of the present invention.

The invention provides a microbial preparation for controlling the transmission of mosquito-borne diseases, including a bacterial carrier, a polypeptide chain, and a mosquito toxin protein, wherein the bacterial carrier, the polypeptide chain and the mosquito toxin protein are different species according to different purposes To make changes, for example, the best embodiment of the present invention uses an environmental microorganism to control the spread of dengue fever, which is spread by mosquitoes and whose larvae are grown in water; and B. subtilis is commonly used. In order to adjust the environmental probiotics of the water quality, the environmental probiotics use B. subtilis . The polypeptide chain selects an amino acid sequence with a cleavable position that can be digested by mosquito intestinal enzymes. The mosquito toxin protein is selected. Mosquito killing protein.

As shown in FIG. 1, the present invention aims to establish a microbial preparation 10 for controlling the spread of dengue fever, which includes a bacterial carrier 101, a polypeptide chain 103 that can be digested by the enzymes of the intestinal tract, and a mosquito poisoning protein 104, wherein, the The mosquito toxin 104 is expressed on the membrane binding protein 102 of the bacterial carrier 101 through the polypeptide chain 103. When 孑孓20 eats from the environment, it will ingest the microbial preparation of the present invention together. After the intestinal enzyme 201 decomposes the polypeptide chain 103, the mosquito-toxin 104 will be released to achieve mosquito killing. The purpose of controlling the spread of dengue fever.

The embodiment of the microbial preparation according to the present invention is as follows:

1. Construction of E.coli (E.coli) expression system fusion mosquito toxin protein recombinant gene expression plasmid

The present invention first constructs an expression plastid fused with a mosquito toxin protein gene expressed in E. coli , which contains a recombinant gene and an expression plastid for laboratory testing.

The mosquito toxin-killing gene of the recombinant gene of the present invention selects truncated Mosquitocidal Toxin 2 (tMTX2) gene from truncated modified Bacillus sphaericus type 31-2 and clones the recombinant gene (clone) to pET42b expression in vivo. Please refer to FIG. 2, the recombinant gene implanted in the present invention is connected to the GST tag gene at the 5′ end and the AIDA membrane protein gene at the 3′ end, and a segment is designed between the tMTX2 gene and the AIDA gene to express the inclusion of SEQ ID NO.1 gene of the polypeptide chain (GGG-KRWY-GGG), which has the amino acid sequence of the trypsin like protease digested by the mosquito intestine and both ends The short peptide gene used to stabilize the polypeptide chain is called KM4 recombinant gene (Experimental group 1), and a KM6 recombinant gene (experimental) that does not contain the polypeptide chain gene (replace GGG-KRWY-GGG with GGGSG) is also designed. Group 2), and the KM3 recombinant gene (control group) without the polypeptide chain gene and the membrane protein gene.

2. Recombinant protein expression of E. coli expression system fusion mosquito toxin protein

Transform KM3, KM4 and KM6 recombinant gene fusion mosquito toxin protein expression plasmids into BL21 (DE3) type E. coli expression vectors respectively, and incubate with LB medium until the bacterial concentration absorbance reaches OD600nm At about 0.4, start adding 0.1 mM isopropyl-β-D-thiogalactoside (Isopropyl β-D-1-thiogalactopyranoside; IPTG) induction culture to perform protein expression and produce KM3 and KM4 And KM6 recombinant protein. Because the protein sizes of KM3, KM4 and KM6 are different, the induction time is KM3 induction for 0.5 hours, KM4 induction for 3 hours and KM6 induction for 1 hour, in order to achieve the same protein expression. Afterwards, SDS-PAGE and Western blotting were used to verify the protein. As shown in Figure 3, the left picture shows the results of SDS-PAGE, and the right picture shows the results of Western blotting with mouse-derived primary antibodies against GST tag. . The results showed that the protein sizes of KM4 (SEQ ID NO.3) and KM6 (SEQ ID NO.4) were the expected 108kDa (kilo-Dalton), while the protein sizes of KM3 (SEQ ID NO.2) were Expected 58kDa.

3. Toxicity test of BL21(DE3) type E.coli (E.coli) expressed by tMTX2-AIDA against Egyptian zebra larvae

After the protein performance was confirmed, KM4 and KM6 were used for the cytotoxicity test of Egyptian spotted larvae. As shown in FIG. 4A, KM4 has the polypeptide chain of the present invention, and its tMTX2 can be cleaved and separated by mosquito intestinal enzymes. tMTX2 cannot be cleaved and separated by mosquito gut enzymes.

Shown, KM4 KM6 group and groups with different concentrations in FIG. ^{4b (10 5 cells / ml-} 10 8 cells / ml) cultured with Egyptian markings larvae 60 hours (n = 5, and independent experiment was repeated three times) was measured after The mortality rate, and the untransformed BL21 (DE3) type E. coli (also at a concentration of OD600nm about 0.4, IPTG induced culture for 3 hours) as a control group. The results show that compared with the control group, KM4 and KM6 have obvious toxin killing effect, and the toxin killing effect of KM4 is faster and more effective than KM6, and the half lethal dose (LC50) of the death caused by it is about 10 ⁵ Cells/ml-10 ⁶ cells/ml, this result proves that this strategy is successful in E. coli system.

4. Construction of B. subtilis (B. subtilis) expression system fusion mosquito toxin protein recombinant gene expression plasmid

After the E. coli expression system proves that the present invention is successful, the present invention is further implemented in environmental probiotics, wherein the selected environmental probiotic is B. subtilis .

The recombinant gene of the present invention is constructed in the same way as the E. coli expression system, and a segment is designed between the mosquito killing protein (tMTX2) and the membrane binding protein to express the polypeptide chain (GGG) containing SEQ ID NO.1 -KRWY-GGG) gene, the difference is that the recombinant gene used in this time is connected with an additional HA tag gene before the membrane binding protein gene. Because B. subtilis was selected as the bacterial carrier, the membrane-binding protein gene was selected as the YhcR gene. As shown in Figure 5, two different recombinant genes were constructed in this experiment, which were named recombinant genes of Bacillus subtilis expression system a and Bacillus subtilis expression system recombinant gene b, wherein the Bacillus subtilis expression system recombinant gene a can express "(tMTX 2)-(GGG-KRWY-GGG)-(HA)-(YhcR)" protein (SEQ ID NO. 5), the recombinant gene b of Bacillus subtilis expression system is added with a GST tag at the 5'end, which can express "(GST)-(tMTX 2)-(GGG-KRWY-GGG)-(HA)-(YhcR)" Protein (SEQ ID NO. 6). Both are cloned into pHY300PLK expression plasmid.

5. Bacillus subtilis (B. subtilis) performance fusion mosquito toxin protein

Transform the mosquito-toxin protein-expressing plastids of recombinant gene a of Bacillus subtilis expression system and recombinant gene b of Bacillus subtilis expression system into B. subtilis vector for expression, and cultivate in LB culture solution to the bacterial concentration to absorb light When the value reaches OD600nm of about 0.6, 1 mM isopropyl-β-D-1-thiogalactopyranoside (Isopropyl β-D-1-thiogalactopyranoside; IPTG) induction culture is started for 3 hours, and protein expression is performed. Afterwards, the protein was verified by SDS-PAGE and Western blot method. The two different recombinant genes in this experiment, the recombinant protein a of Bacillus subtilis expression system, translated from recombinant gene a of Bacillus subtilis expression system, the total length of the recombinant protein a was 56kDa, and Bacillus subtilis The full-length recombinant protein b of Bacillus subtilis expression system translated from expression system recombinant gene b has a total length of 80 kDa. As shown in Figure 6, the Western blot method using a primary antibody against HA tag showed that the recombinant protein a of Bacillus subtilis expression system did not produce the expected 56kDa protein, but many defective proteins (about 36kDa) appeared. The expression protein size of the recombinant protein b of the Bacillus subtilis expression system is 80 kDa, which proves that the recombinant protein b line of the Bacillus subtilis expression system is relatively stable and successfully expressed.

From the foregoing results, it can be confirmed that the expression of tMTX2 protein through the polypeptide chain of the present invention on the membrane binding protein of the bacterial carrier does have a good efficiency of killing mosquito larvae, and the present invention also successfully expressed the tMTX2 protein in Bacillus subtilis ( B. subtilis ), the microbial preparation of the present invention can be used for improving water quality and controlling the spread of mosquito-borne diseases.

<110> Kaohsiung Medical University (Kaohsiung Medical University) Cheng, Tian-Lu Huang, Chien-Chiao Lin, Wen-Wei

<120> A MICROBIAL AGENT FOR CONTROLLING TRANSMISSION OF MOSQUITO-BORNE DISEASES

<130> 3098-KMU-TW

<160> 6

<170> PatentIn version 3.5

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10‧‧‧Microbiological preparation for controlling the transmission of mosquito-borne diseases

101‧‧‧ Bacteria carrier

102‧‧‧ Membrane binding protein

103‧‧‧ polypeptide chain

104‧‧‧ Mosquito poison protein

20‧‧‧

201‧‧‧The intestinal enzymes

Claims (18)

A polypeptide chain includes: an amino acid sequence with trypsin like protease cleavage for mosquito intestinal digestion; and short peptides at both ends for stabilization of the amino acid sequence. The polypeptide chain according to claim 1, wherein the amino acid sequence includes at least monoamine acid (K) or monoarginine (R). The polypeptide chain of claim 1, wherein the amino acid sequence comprises the sequence of SEQ ID NO.1. A microbial preparation for controlling the spread of mosquito-borne diseases, including: a bacterial carrier; as in claim 1, the polypeptide chain is attached to the cell membrane of the bacterial carrier; and a mosquito toxin protein, which is attached to the polypeptide chain It is expressed on the bacterial carrier. The microbial preparation according to claim 4, wherein the bacterial carrier is an environmental probiotic. The microbial preparation according to claim 4, wherein the environmental probiotic bacteria include bacteria of the genus Bacillus , photosynthetic bacteria, and nitrifying bacteria. The microbial preparation according to claim 4, wherein the amino acid sequence includes at least monoamine acid (K) or monoarginine (R). The microbial preparation according to claim 4, wherein the amino acid sequence comprises the sequence of SEQ ID NO.1. The microbial preparation according to claim 4, wherein the mosquito toxin protein includes a first type mosquito killing protein (Mosquitocidal Toxin 1), a second type mosquito killing protein (Mosquitocidal Toxin 2), a third type mosquito killing protein (Mosquitocidal Toxin 2) Toxin 3), Cry protein (Cry family proteins), Cyt protein (Cyt family proteins), CLP protein (Cyclic lipopeptide protein), or any combination thereof. The microbial preparation according to claim 4, wherein the mosquito toxin is Mosquitocidal Toxin 2 (Mosquitocidal Toxin 2). The microbial preparation according to claim 4, wherein the mosquito toxin is truncated modified mosquito killer protein (truncated Mosquitocidal Toxin 2). The microbial preparation according to claim 4, wherein the polypeptide chain is attached to a cell membrane binding protein on the cell membrane of the bacterial carrier. A recombinant gene, comprising: a cell membrane binding protein gene transcribed into a cell membrane binding protein on the cell membrane of the bacterial vector as described in claim 12; a polypeptide chain gene transcribed into a polypeptide as described in claim 1 Chain; and a mosquito toxin protein gene transcribed into the mosquito toxin protein as described in claim 4; wherein, the polypeptide chain gene is disposed between the cell membrane binding protein gene and the mosquito protein gene. The recombinant gene according to claim 13, wherein the polypeptide chain gene includes at least the gene of monoamine (K) or arginine (R). The recombinant gene according to claim 13, wherein the polypeptide chain gene includes a gene that can express the sequence of SEQ ID NO.1. The recombinant gene according to claim 13, wherein the mosquito toxin-killing gene line includes a first type mosquito-killing protein (Mosquitocidal Toxin 1) gene, a second type mosquito-killing protein (Mosquitocidal Toxin 2) gene, a third type mosquito-killing protein Protein (Mosquitocidal Toxin 3) gene, Cry protein (Cry family proteins) gene, Cyt protein (Cyt family proteins) gene, CLP protein (Cyclic lipopeptide protein) gene, or any combination thereof. The recombinant gene according to claim 13, wherein the mosquito toxin protein gene is a Mosquitocidal Toxin 2 gene. The recombinant gene according to claim 13, wherein the mosquito toxin gene is a truncated Mosquitocidal Toxin 2 gene after truncation modification.

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