TWI728264B - 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 agents for controlling the spread of mosquito-borne diseases

The present invention relates to a microbial preparation, in particular to a microbial preparation used to control the spread of mosquito-borne diseases.

Taiwan, China, Southeast Asian countries or other countries in tropical and subtropical climate zones are prone to spread of mosquito-borne diseases such as Japanese encephalitis, Zika virus or dengue fever due to the ravages of mosquitoes. Take dengue fever as an example. China has listed dengue fever as the second type of infectious disease in 1993. In the past 10 years, there have been 800 to 10,000 confirmed cases of dengue fever each year. In 2015, dengue fever The number of confirmed cases has risen to more than 40,000. Although the current medical treatment is advanced and the policy is implemented properly, the number of dengue fever cases is gradually decreasing. However, with the impact of global warming, the areas where mosquitoes can thrive are gradually spreading. According to the statistical table announced by the Control Department, since 2017, the number of confirmed cases of dengue fever in the greater Taipei area has begun to be higher than that in Tainan and Kaohsiung.

Most methods of repelling mosquitoes to the environment use large-scale spraying methods. One is that insecticides are chemical products, which have a great impact on the environment; for the two, larvae grow well in water, and spraying insecticides indirectly pollutes water quality. The quality of domestic water and health effects are worrying, and the development of environmentally friendly methods to kill mosquitoes is a top priority. At present, the whole world is committed to biological control work, advocating the relative relationship between the food chain of organisms and natural enemies to control pests. The present invention uses the concept of food chain to design a food chain that allows larvae to be ingested and ingest mosquito toxic proteins. And achieve the effect of killing mosquitoes.

Past studies have pointed out that Bacillus thuringiensis and Bacillus sphaericus of the genus Bacillus have mosquito-toxin proteins that can kill mosquitoes. There are also some insecticides made by Bacillus thuringiensis, but their The disadvantages are 1. Bacillus thuringiensis is not an environmental probiotic, 2. Bacillus thuringiensis only has mosquito-killing properties during the sporulation stage, and 3. Bacillus thuringiensis preparations themselves have no ability to replicate. Therefore, the present invention provides a new idea Using a polypeptide chain that can be digested by larval intestinal enzymes, the mosquito virulence protein is expressed on the membrane-bound protein of the cell membrane of environmental probiotics, and the bacterial carrier has the characteristics of self-proliferation in the environment, so that the insecticide has Reproducibility, and the mosquito toxic protein can be released faster and more efficiently.

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

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

The purpose of the present invention is to provide an environmentally friendly novel microbial preparation for controlling the spread of mosquito-borne infectious diseases. The main concept is to use an environmental microorganism as a carrier and express a mosquito toxic 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 a trypsin like protease cut position for mosquito intestinal digestion, and short peptides at both ends to stabilize The amino acid sequence. Wherein, the amino acid sequence includes at least one lysine (K) or one arginine (R).

Another main technical feature of the present invention is a microbial preparation for controlling the spread of mosquito-borne diseases, which uses the polypeptide chain to connect the mosquito virulence protein to the cell membrane of the bacterial carrier. It includes a bacterial carrier, a polypeptide chain, and a mosquito venom Protein, wherein the mosquito-killing protein refers to the mosquito-killing protein.

According to the microbial preparation of the present invention, in a preferred embodiment, the bacterial carrier system is an environmental probiotic, including bacteria of the genus Bacillus , 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 at least includes a lysine (K) or an arginine (R).

According to the microbial preparation of the present invention, in a preferred embodiment, the mosquito-toxin protein includes the first type mosquito-killing protein (Mosquitocidal Toxin 1), the second type mosquito-killing protein (Mosquitocidal Toxin 2), and the 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 toxic protein is Mosquitocidal Toxin 2 (Mosquitocidal Toxin 2), wherein when the mosquito toxic protein is truncated and modified, the mosquitocidal protein is a modified second type mosquito The effect is better when protein (truncated Mosquitocidal Toxin 2) is used.

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

The main technical feature of the present invention also includes a recombinant gene, including a membrane-associated protein gene, a polypeptide chain gene, and a mosquito-killing protein gene that can be transcribed from the microbial preparation, wherein the polypeptide chain gene is set in Between the cell membrane binding protein gene and the mosquito virulence protein gene.

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

According to the recombinant gene of the present invention, in a preferred embodiment, the gene of the mosquito virulence protein includes the first type mosquitoicidal protein (Mosquitocidal Toxin 1) gene, the second type mosquitoicidal protein (Mosquitocidal Toxin 2) gene, and the second type mosquitoicidal protein (Mosquitocidal Toxin 2) gene. Mosquitocidal Toxin 3 gene, Cry family proteins gene, Cyt family proteins 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 mosquitocidal protein is a type 2 mosquito-killing protein (Mosquitocidal Toxin 2) gene, wherein, when the mosquitocidal toxin protein is truncated and modified, the mosquitocidal-toxin protein is a modified type 2 mosquito-killing protein. The effect is better when the mosquito protein (truncated Mosquitocidal Toxin 2) gene is used.

According to the microbial preparation of the present invention, in a preferred embodiment, the membrane-associated protein gene can be selected according to different bacterial vectors. For example, on E. coli , the membrane-associated protein gene can be selected Select AIDA protein gene or OmpA protein gene; if it is on B. subtilis (B. subtilis ), the cell membrane binding protein can select YhcR protein gene.

The microbial preparation of the present invention is used to control mosquito-borne diseases by administering an appropriate amount of the present invention in water. Since mosquito larvae-larvae grow in water, especially stagnant water, an appropriate amount of the present invention The microbial preparation of the present invention is thrown into water, and the larvae will take in the microbial preparation of the present invention while eating. After the polypeptide chain is decomposed by its intestinal enzymes, the mosquito toxic protein is released, thereby killing mosquitoes and controlling mosquitoes. The purpose of vector-borne diseases.

10‧‧‧Microbial agents for controlling the spread of mosquito-borne diseases

101‧‧‧Bacterial carrier

102‧‧‧cell membrane binding protein

103‧‧‧polypeptide chain

104‧‧‧Mosquito toxic protein

20‧‧‧ larva

201‧‧‧The larval intestinal enzymes

Fig. 1 is a diagram of an implementation aspect of the present invention.

Figure 2 is the recombinant genome of the fusion mosquito virulence protein gene used in the E. coli expression system test of the present invention. The KM4 recombinant gene is the recombinant gene experimental group 1 of the present invention; the KM6 recombinant gene does not contain the polypeptide chain gene ( The experimental group 2 replaced by GGGSG; the KM3 recombinant gene is the control group without the polypeptide chain gene and the cell membrane protein gene.

Figure 3 shows the results of the recombinant protein expression of the fusion mosquito virulence protein used in the E. coli expression system test of the present invention. The left picture is the result of SDS-PAGE, and the right picture is the result of a mouse-derived anti-GST tag primary antibody. The result of Western ink dot method.

Fig. 4 is the result of the toxicity of the present invention on the E. coli expression system against Egyptian streaked larvae. Fig. A is the implementation diagram of KM4 and KM6 entering the larvae. Fig. B is the result of the toxicity test of KM4 and KM6. .

Fig. 5 is a recombinant gene of B. subtilis expression system fused with mosquito venom protein used in B. subtilis performance test of the present invention.

Figure 6 shows the results of the recombinant protein expression of the B. subtilis expression system fused with mosquito venom protein used in the performance test of B. subtilis according to the present invention.

The present invention provides a microbial preparation for controlling the spread of mosquito-borne infectious diseases, comprising a bacterial carrier, a polypeptide chain, and a mosquito toxic protein, wherein the bacterial carrier, the polypeptide chain and the mosquito toxic protein are of different species according to different purposes To be modified, for example, the best embodiment of the present invention uses an environmental microorganism to control the spread of dengue fever, and dengue fever is transmitted by mosquitoes, whose larvae are larvae that grow in water; and, B. subtilis (B. subtilis ) is commonly used To adjust the water quality of the environmental probiotics, the environmental probiotics are selected from B. subtilis (B. subtilis ), the polypeptide chain is selected from an amino acid sequence that can be digested by mosquito intestinal enzymes, and the mosquito toxic protein is selected Mosquito-killing protein.

As shown in Figure 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 larval intestinal enzymes, and a mosquito venom protein 104, wherein the The mosquito virulence protein 104 is expressed on the membrane-bound protein 102 of the bacterial carrier 101 by the polypeptide chain 103. When the larva 20 eats from the environment, the microbial preparation of the present invention will be ingested together. After the larvae’s intestinal enzyme 201 decomposes the polypeptide chain 103, the mosquito toxic protein 104 is subsequently released to achieve mosquito killing effect. The purpose of controlling the spread of dengue fever.

According to the microbial preparation of the present invention, its implementation is as follows:

1. Construct the expression plastid of the fusion mosquito virulence protein recombinant gene of E. coli expression system

The present invention first constructs an expression plastid fused with a mosquito virulence protein gene that can be expressed in Escherichia coli (E. coli ), which includes a recombinant gene and an expression plastid to facilitate laboratory experiments.

The mosquito toxic protein gene of the recombinant gene of the present invention selects the truncated Mosquitocidal Toxin 2 (tMTX2) gene derived from the 31-2 type Bacillus sphaericus after truncation and modification, and clones the recombinant gene (clone) to pET42b expression in plastids. Please refer to Figure 2, the recombinant gene implanted in the present invention, the 5'end is connected to the GST tag gene, the 3'end is connected to the AIDA membrane protein gene, and a section is designed between the tMTX2 gene and the AIDA gene to show that it contains SEQ ID NO.1 polypeptide chain (GGG-KRWY-GGG) gene, the polypeptide chain gene has an amino acid sequence cut by trypsin like protease that can be digested by mosquito intestines and both ends The short peptide gene used to stabilize the polypeptide chain is called the KM4 recombinant gene (experimental group 1). At the same time, a KM6 recombinant gene that does not contain the polypeptide chain gene (GGG-KRWY-GGG is replaced by GGGSG) (experimental) Group 2), and KM3 recombinant gene without the polypeptide chain gene and cell membrane protein gene (control group).

2. Recombinant protein expression of the fusion mosquito virulence protein of E. coli expression system

Transform the fusion mosquito protein expression plastids of KM3, KM4 and KM6 recombinant genes into BL21 (DE3) E. coli expression vectors, and culture them in LB medium until the absorbance value of the bacteria reaches OD600nm At about 0.4, start to add 0.1mM isopropyl-β-D-thiogalactopyranoside (Isopropyl β-D-1-thiogalactopyranoside; IPTG) induction culture, perform protein expression, and produce KM3, KM4 And KM6 recombinant protein. Because the protein sizes of KM3, KM4 and KM6 are different, KM3 is induced for 0.5 hours, KM4 is induced for 3 hours, and KM6 is induced for 1 hour, respectively, in order to achieve consistent protein expression. Afterwards, protein verification was performed by SDS-PAGE and Western blotting method, as shown in Figure 3. The left picture is the result of SDS-PAGE, and the right picture is the result of Western blotting method with the mouse-derived anti-GST tag primary antibody. . The results showed that the expressive protein size of KM4 (SEQ ID NO. 3) and KM6 (SEQ ID NO. 4) was the expected 108kDa (kidalton), while the expressive protein size of KM3 (SEQ ID NO. 2) was The expected 58kDa.

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

After the protein expression was confirmed, KM4 and KM6 were used to test the toxicity of Egyptian spotted larvae respectively. As shown in Figure 4A, KM4 has the polypeptide chain of the present invention, and its tMTX2 can be cut and separated by mosquito intestinal enzymes. tMTX2 cannot be cut and separated by mosquito intestinal enzymes.

As shown in Figure 4b, the KM4 group and KM6 group were ^{cultured with Egyptian streaked larvae at different concentrations (10 5} cells/ml-10 ⁸ cells/ml) for 60 hours (n=5, independent and repeated experiments), and then measured Its mortality, and the untransformed BL21(DE3) E. coli (also at a concentration of OD600nm of about 0.4, cultured with IPTG for 3 hours) was used as a control group. The results show that compared with the control group, KM4 and KM6 have obvious poisonous effects, and the poisonous effect of KM4 is faster and more effective than that of KM6, and its half lethal dose (LC50) for larval death is about 10 ⁵ Cells/ml-10 ⁶ cells/ml or so, this result proves that this strategy is successful on the E. coli system.

4. Construct the expression plastid of the fusion mosquito virulence protein recombinant gene of B. subtilis expression system

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 (B. subtilis ).

The recombinant gene of the present invention is constructed in the same way as the E. coli expression system. 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 this time is connected to a HA tag gene before the membrane binding protein gene. Since B. subtilis was used as the bacterial carrier, the YhcR gene was selected for the membrane-associated protein gene. As shown in Figure 5, two different recombinant genes were constructed in this experiment and named as B. subtilis expression system recombinant genes. a and Bacillus subtilis expression system recombinant gene b. Among them, the Bacillus subtilis expression system recombinant gene a can express "(tMTX 2)-(GGG-KRWY-GGG)-(HA)-(YhcR)" protein (SEQ ID NO. 5), Bacillus subtilis expression system recombinant gene b is to add a GST tag at the 5'end, which can express "(GST)-(tMTX 2)-(GGG-KRWY-GGG)-(HA)-(YhcR)" The protein (SEQ ID NO. 6). Both are cloned into pHY300PLK expression plastids.

5. B. subtilis (B. subtilis) expresses fusion mosquito toxic protein

The fusion mosquito protein expression plastids of B. subtilis expression system recombinant gene a and B. subtilis expression system recombinant gene b were transformed into B. subtilis vectors for expression, and cultured in LB medium to absorb light at the bacterial concentration When the value reaches OD600nm is about 0.6, start to add 1mM isopropyl-β-D-thiogalactopyranoside (Isopropyl β-D-1-thiogalactopyranoside; IPTG) induction (induction) culture for 3 hours for protein expression. Afterwards, the protein was verified by SDS-PAGE and Western blotting method. The two different recombinant genes in this experiment, the Bacillus subtilis expression system recombinant gene a, were translated from the Bacillus subtilis expression system recombinant protein a. The total length of the recombinant protein a is 56kDa, Bacillus subtilis The total length of the Bacillus subtilis expression system recombinant protein b translated from the expression system recombinant gene b is 80kDa. As shown in Figure 6, the Western blot method with the mouse-derived primary antibody against HA tag showed that the recombinant protein a of the Bacillus subtilis expression system did not produce the expected 56kDa protein, instead there were many defective proteins (about 36kDa). The size of the recombinant protein b of the Bacillus subtilis expression system was the expected 80kDa, which proved that the recombinant protein b of the Bacillus subtilis expression system was relatively stable and successfully expressed.

It can be confirmed from the foregoing results that the expression of tMTX2 protein on the membrane-bound protein of the bacterial carrier through the polypeptide chain of the present invention does have a good efficiency in 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 to improve water quality and control the spread of mosquito-borne diseases at the same time.

<110> Kaohsiung Medical University (Kaohsiung Medical University)

<120> Microbial preparations to control the spread of mosquito-borne diseases

<130> 3098-KMU-TW

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<170> PatentIn version 3.5

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10‧‧‧Microbial agents for controlling the spread of mosquito-borne diseases

101‧‧‧Bacterial carrier

102‧‧‧cell membrane binding protein

103‧‧‧polypeptide chain

104‧‧‧Mosquito toxic protein

20‧‧‧ larva

201‧‧‧The larval intestinal enzymes

Claims (13)

A polypeptide chain for mosquito larval intestinal enzyme decomposition, which is composed of an amino acid sequence of SEQ ID NO. 1 and short peptides used to stabilize the amino acid sequence at both ends, wherein the two ends are used to stabilize the amino acid sequence. The short peptide of amino acid sequence is GGG. A microbial preparation for controlling the spread of mosquito-borne infectious diseases, comprising: a bacterial carrier with at least one membrane-binding protein on the cell membrane, and the bacterial carrier is not Bacillus thuringiensis; a polypeptide for mosquito larval intestinal degradation The chain is composed of an amino acid sequence of SEQ ID NO. 1 and a short peptide GGG whose two ends are used to stabilize the amino acid sequence, and the two ends are used to stabilize the amino acid sequence. Wherein one end of the polypeptide chain is joined to the at least one membrane-bound protein of the cell membrane of the bacterial carrier for decomposition of mosquito larvae intestinal enzymes; and a mosquito toxic protein, which is different from the one by being joined to the polypeptide chain The other end of at least one membrane-bound protein is expressed on the bacterial carrier. The microbial preparation according to claim 2, wherein the bacterial carrier system is an environmental probiotic. The microbial preparation according to claim 3, wherein the environmental probiotics include bacteria of the genus Bacillus , photosynthetic bacteria and nitrifying bacteria. The microbial preparation according to claim 2, wherein the mosquito toxic protein system includes Mosquitocidal Toxin 1 (Mosquitocidal Toxin 1), Mosquitocidal Toxin 2 and Mosquitocidal Toxin 2 (Mosquitocidal Toxin 2) and Mosquitocidal Toxin 2 (Mosquitocidal Toxin 2), Mosquitocidal Toxin 2 (Mosquitocidal Toxin 2), Mosquitocidal Toxin 2 (Mosquitocidal Toxin 2), Mosquitocidal Toxin 2 (Mosquitocidal Toxin 2) and Mosquitocidal Toxin 2 (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 2, wherein the mosquito toxic protein is Mosquitocidal Toxin 2 (Mosquitocidal Toxin 2). The microbial preparation according to claim 2, wherein the mosquito toxic protein is truncated Mosquitocidal Toxin 2 (truncated Mosquitocidal Toxin 2). The microbial preparation according to claim 2, wherein the bacterial carrier system is selected from Escherichia coli or Bacillus subtilis, and when the bacterial carrier system is E. coli, the at least one membrane-bound protein is AIDA protein; when the bacterial carrier system In the case of Bacillus subtilis, the at least one membrane-bound protein is YhcR protein. A recombinant gene, comprising: a membrane binding protein gene translated into the at least one membrane binding protein on the cell membrane of the bacterial vector as described in claim 2, and the bacterial vector is not Bacillus thuringiensis; A polypeptide chain gene translated into the polypeptide chain as described in claim 2; and a mosquito venom protein gene translated into the mosquito venom protein as described in claim 2; wherein the polypeptide chain gene is set at the at least Between a cell membrane binding protein gene and the mosquito-killing protein gene. The recombinant gene according to claim 9, wherein the mosquito virulence protein gene line includes a type 1 mosquito killing protein (Mosquitocidal Toxin 1) gene, a type 2 mosquito killing protein (Mosquitocidal Toxin 2) gene, and a type 3 mosquito killing protein gene. Mosquitocidal Toxin 3 gene, Cry family proteins gene, Cyt family proteins gene, CLP protein (Cyclic lipopeptide protein) gene or any combination thereof. The recombinant gene according to claim 9, wherein the mosquito virulence protein gene is a Mosquitocidal Toxin 2 (Mosquitocidal Toxin 2) gene. The recombinant gene according to claim 9, wherein the mosquito toxic protein gene is a truncated Mosquitocidal Toxin 2 (truncated Mosquitocidal Toxin 2) gene after modification. The recombinant gene according to claim 9, wherein the bacterial carrier system is selected from Escherichia coli or Bacillus subtilis, and when the bacterial carrier system is Escherichia coli, the at least one membrane-associated protein is the gene of the AIDA protein; when the bacteria When carrying Bacillus subtilis, the at least one membrane-binding protein is the gene of YhcR protein.

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