阅读说明：本技术 一种抗贾第虫作用靶点的锌脂蛋白及医用用途 (Zinc lipoprotein with anti-giardia effect target and medical application ) 是由 张西臣 郑敬彤 李建华 宫鹏涛 张楠 李新 王晓岑 杨举 马赫然 于 2021-01-04 设计创作，主要内容包括：本发明提供了一种抗贾第虫作用靶点的锌脂蛋白及医用用途,发现了能显著抑制虫体繁殖的蛋白ZFD,其通过作用于贾第虫TRBD抑制贾第虫的繁殖,降低端粒酶活性以及影响端粒长度；本发明为贾第虫的治疗提供了新的靶点ZFD,为未来药物干预贾第虫病奠定基础。新的端粒酶相关蛋白,找出针对端粒酶的作用靶点是抗贾第虫药物开发的新途径。(The invention provides a zinc lipoprotein with an anti-giardia effect target point and medical application, and discovers a protein ZFD capable of obviously inhibiting propagation of giardia, which can inhibit the propagation of giardia by acting on giardia TRBD, reduce telomerase activity and influence telomere length; the invention provides a new target ZFD for the treatment of giardia, and lays a foundation for the future medicine intervention of giardia. The novel telomerase related protein finds out the action target aiming at the telomerase, and is a novel way for developing anti-giardia medicaments.)

1. A giardia zinc lipoprotein, which has the gene sequence as follows: shown in SEQ No 1, the molecular formula is: c₉₂₃H₁₄₃₈N₂₇₄O₂₉₅S₁₅The relative molecular mass was 21.6KD, and the isoelectric point was 6.70.

2. The giardia zinc lipoprotein of claim 1 as a target for the action of anti-giardia drugs.

3. The medical use of giardia zinc lipoprotein of claim 1 in the preparation of an anti-giardia drug.

Technical Field

The invention provides zinc lipoprotein serving as a new anti-giardia effect target point, which can be used for developing anti-giardia medicaments and belongs to the technical field of antiparasitic.

Background

Giardia duodenalis: (Giardia duodenalis) Giardia (Giardia), as a zoonotic parasite, is predominantly parasitic in the small intestine of humans and some mammals, causing giardiasis, which is characterized primarily by diarrhea, and severely harming the health of humans and other animals. Controlling giardiasis still mainly relies on the use of anti giardiac medicine at present, if: metronidazole, etc., but there are no ideal drugs and vaccines for controlling giardiasis to date due to the development of drug resistance. As giardia can be repeatedly passaged in vitro to generate immortalization, the regulation and control of giardia are closely related to telomeres. Telomeres are a special nucleoprotein complex, are located at the end of eukaryotic cell chromosomes, play an important role in chromosome location, replication, protection and control of cell growth and life, and are closely related to apoptosis, cell transformation and immortalization. Telomerase is an enzyme responsible for lengthening telomeres, can add telomere DNA to the end of chromosome of eukaryotic cells, and plays an important role in keeping telomeres stable, complete genome, long-term activity of cells and the like. The regulation of telomerase activity is related to telomerase relative protein.

Disclosure of Invention

The invention aims to provide an anti-giardia target point which can regulate and control the activity of giardia telomerase and further play a role in anti-giardia.

The invention discloses a giardia zinc lipoprotein (the following abbreviation: ZFD), the gene sequence of which is as follows: shown in SEQ No 1, the molecular formula is: c₉₂₃H₁₄₃₈N₂₇₄O₂₉₅S₁₅The relative molecular mass is 21.6KD, and the isoelectric point is 6.70; 3D structure prediction was performed on ZFD using SWISS-MODEL, the results are shown in FIG. 1.

The giardia zinc lipoprotein provided by the invention is used as a target point of the anti-giardia drug action.

The giardia zinc lipoprotein provided by the invention has medical application in preparing anti-giardia medicines.

The giardia zinc lipoprotein target of the invention is an interaction protein of a telomerase reverse transcriptase (TERT) middle Telomerase RNA Binding Domain (TRBD) structural domain, and a ribozyme transferred into zinc lipoprotein can obviously reduce the expression of the zinc lipoprotein and has obvious influence on the reproduction of the worm body. The TRAP method is adopted to detect the influence of ribozyme on the giardia telomerase activity, and the result shows that the expression level of zinc lipoprotein is reduced, and the giardia telomerase activity is down-regulated.

According to the invention, the RT-PCR method is adopted to detect the telomere length of the Giardia ribozyme experimental group and the control group, and the result shows that the telomere length is lower than that of the negative control group, which indicates that the reduction of the ZFD expression level influences the telomere length.

The invention has the positive effects that:

disclosed is a novel giardia zinc lipoprotein, which finds a protein ZFD that can significantly inhibit the reproduction of giardia, reduces telomerase activity and affects telomere length by acting on giardia TRBD to inhibit the reproduction of giardia; the invention provides a new target ZFD for the treatment of giardia, and lays a foundation for the future medicine intervention of giardia. The novel telomerase related protein finds out the action target aiming at the telomerase, and is a novel way for developing anti-giardia medicaments.

Drawings

FIG. 1 3D structure prediction plots of ZFD proteins of the invention;

FIG. 2 the inhibitory effect of the ZFD ribozymes of the present invention on Giardia telomerase activity (M: DNA molecular weight (DL500 DNA Marker); 1: HeLa cell TRAP product (control); 2: transfection of normal Giardia control group; 3: transfection of Giardia control group containing the empty vector; 4: transfection of Giardia control group containing the pC631-SNAP-Ham-GDH vector; 5: transfection of Giardia group containing the pC631-SNAP-Ham-ZFD vector);

FIG. 3 shows the PCR result of ZFD telomere electrophoresis verification (Giardia telomere PCR result: M: 50 bp DNA Ladder; 1: telomere 1 primer PCR result; 2: telomere 2 primer PCR result; 3: internal reference PCR result);

FIG. 4 shows the inhibitory effect of the ZFD ribozymes of the present invention on the telomere length of Giardia (telomere length detection. Control: normal Giardia trophozoite telomere length; ZFD-sub: Giardia trophozoite telomere length transfected with vector pC 631-SNAP-ZFD-sub; Ham-GDH: Giardia trophozoite telomere length transfected with vector pC 631-SNAP-Ham-GDH; Ham-ZFD: Giardia trophozoite telomere length transfected with vector pC 631-SNAP-Ham-ZFD);

FIG. 5 the effect of the ZFD ribozymes of the invention on the cleavage of ZFD transcripts;

FIG. 6 shows the inhibition effect of ZFD ribozymes of the present invention on Giardia propagation (Giardia body count of transfected ribozymes, control group: uncharged group; group1 group: empty vector electrotransfer group; gourp2 group: negative group; group3 group: ribozyme electrotransfer group).

Detailed Description

The present invention is further illustrated by the following examples, which do not limit the present invention in any way, and any modifications or changes that can be easily made by a person skilled in the art to the present invention will fall within the scope of the claims of the present invention without departing from the technical solution of the present invention.

Example 1

The invention discloses a giardia zinc lipoprotein (ZFD) stabilizing protein, ZFD), the molecular formula is: c₉₂₃H₁₄₃₈N₂₇₄O₂₉₅S₁₅The relative molecular mass is 21.6KD, the isoelectric point is 6.70, the giardia telomerase activity can be regulated and controlled, the giardia resistant effect is further achieved, SWISS-MODEL is used for conducting 3D structure prediction on ZFD, and the gene sequence is as follows: SEQ No. 1, the results are shown in FIG. 1.

Example 2

TRAP detects the effect of ZFD ribozyme on telomerase activity:

after 10 mu L of ZFD ribozyme enters the giardia trophozoite through electrotransfection, a TRAP method is adopted to detect a giardia ribozyme transfection group, a negative control group and an empty vectorTelomerase activity of the group. Washing trophozoite with RNase-free PBS 3 times, and collecting 1 × 10⁷Adding 200 mu L of lysate into each insect body, and grinding for 35 times on ice by using a high-speed tissue grinder for 1-2 sec each time. Performing ice bath lysis for 20min, then 12000g, centrifuging at 4 deg.C for 15min, taking supernatant, measuring protein concentration by BCA method, subpackaging, reacting at 30 deg.C for 30min, and inactivating at 95 deg.C for 5 min. Then, 1. mu.L of PR primer and 1. mu.L of rTaq were added for PCR amplification. The amplification parameters were: 94 ℃ for 1min, 59 ℃ for 1min, 72 ℃ for 1min, 33 cycles. The reaction results were detected by electrophoresis. The results showed that there was no significant difference between the negative control group and the empty vector group, whereas the telomerase activity of the ribozyme-transfected group was lower than that of the negative control group, indicating that the reduction of ZFD expression level down-regulates the giardia's telomerase activity (as shown in figure 2).

Example 3

The RT-PCR method is used for detecting the influence of ZFD ribozyme on telomere length:

extracting Giardia RNA, carrying out reverse transcription to obtain cDNA, respectively designing telomere primers and internal reference according to Giardia telomere DNA repetitive sequence, carrying out PCR amplification, and carrying out PCR amplification at 94 ℃ for 5 min; 30 cycles of 94 ℃ for 30 s, 60 ℃ for 30 s and 72 ℃ for 1min are repeated; 10min at 72 ℃; and cooling to 4 ℃, taking out, and verifying the PCR result by electrophoresis.

In order to detect whether the telomere primer result meets the standard, 5 mu L of PCR product is taken for agarose gel electrophoresis detection after RT-PCR is finished. The result shows that after 30 cycles of amplification, a plurality of bands appear from about 60bp, the length extends to over 500bp, and the telomere detection result meets the expected standard, so that the real-time fluorescence quantitative PCR is a reliable method for determining the telomere length of the giardia genome, and the electrophoresis verifies the PCR result (as shown in figure 3).

And detecting the telomere length of the giardia experimental group and the control group by using an RT-PCR method. Calculating formula according to telomere length: [2Ct (telomeres)/2Ct(s) ] -1 = 2- Δ Ct. The result shows that the relative T/S of the telomere length measured by a Ham-ZFD experimental group Q-PCR is 0.83 +/-0.15; the telomere length measured by PCR of the negative control group is 0.96 +/-0.07 relative to T/S, and the telomere length of the experimental group of 0.93 +/-0.05 is lower than that of the negative control group, which indicates that the reduction of the expression level of ZFD influences the telomere length (as shown in figure 4).

Example 4

Expression of ZFD ribozymes in giardia bodies and effects on bodies:

to assess the effect of ZFDs on giardia growth and reproduction, the ribozyme Ham-ZFDs of the ZFD sequence were designed according to the hammerhead ribozyme design principle. The in vitro cutting result shows that the ribozyme has obvious cutting efficiency on the pC631-SNAP-ZFD in vitro transcript RNA of the ZFD gene, which reaches 84 percent, while the pC631-SNAP-ZFD-sub in vitro transcription RNA in the negative control group has small influence on the pC631-SNAP-ZFD, the efficiency is only 2 percent, and the pC631-SNAP-Ham-GDH in the other negative control group has no visible influence on the pC631-SNAP-ZFD (as shown in the attached figure 5).

When trophozoites were in log phase, worms were collected and washed 3 times with PBS. Centrifuging at 2000r/min at normal temperature for 10min, and discarding the supernatant. The trophozoites were resuspended and counted using pre-cooled Cytomix shock buffer solution to a cell concentration of 10⁶-10⁷One/ml and transferred into a cuvette. Ribozyme 10 μ L was shock transfected into giardia trophozoites. After electric shock, the cells were incubated in ice for 15min, and then transferred to giardia culture flasks for culture. Cells were harvested after 24h, untransfected and transfected worms were counted for 5 consecutive days, and the data obtained were statistically analyzed using SPSS software. The result shows that after 5 days of transfection of the giardia trophozoite with the ZFD ribozyme, the number of the giardia trophozoite is only 41% of that of a control group, and the negative control group has small influence, which indicates that the transfer of the ribozyme has obvious inhibition effect on the propagation of the giardia trophozoite (as shown in figure 6).

By combining the experimental results, the giardia zinc lipoprotein target disclosed by the invention acts on parasite TRBD protein, reduces telomerase activity, influences telomere length, exerts an anti-giardia reproduction effect, and can be used for drug research and development in the anti-parasite technical field.

Sequence listing

<110> Jilin university

<120> zinc lipoprotein with anti-giardia effect target and medical application thereof

<160> 1

<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Giardia (Giardia)

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