CN1673352A - A novel bacillus and separating process and application - Google Patents
A novel bacillus and separating process and application Download PDFInfo
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- CN1673352A CN1673352A CNA2005100492399A CN200510049239A CN1673352A CN 1673352 A CN1673352 A CN 1673352A CN A2005100492399 A CNA2005100492399 A CN A2005100492399A CN 200510049239 A CN200510049239 A CN 200510049239A CN 1673352 A CN1673352 A CN 1673352A
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- zucineum
- leukemibacterium
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Abstract
The present invention relates to one new kind of bacilli and its separation process and application. The new bacillus is named as Leukemibacterium zucineum, and has the preservation number of CCTCC M 205004. The new bacillus can induce apoptosis of several kinds of tumors and has application foreground in treating tumors. The new bacillus can enter eukaryotic cell and survive inside eukaryotic cell for long term, so that it may be used as carrier to carry any kind of compound into cell. The new bacillus may be used in preparing tumor treating products and may find its wide use in biomedicine.
Description
Technical field
The present invention relates to a kind of novel microorganism and application thereof, relate in particular to a kind of novel bacterial that separation and Culture obtains from people's tumor cell line K562 cell.
Background technology
K562 clone derives from a slow grain leukaemic's of 53 years old leukemia cell, is built at 1975 (Blood.45 (3): 321-34,1975) by Lozzio CB and Lozzio BB to be.This cell is the Philadelphia chromosome positive, is a leukemia cell system that worldwide is widely used.
At present, do not appear in the newspapers about in people's K562 cell, extracting and be separated to bacterium; We are separated to a kind of bacterium from this clone, this bacterium and human cell have very good symbiotic relationship, and this also is that the past is without any report.This shows that this bacterium is the mankind's isolating novel bacterials from nature.
Summary of the invention
An object of the present invention is to provide a kind of new bacterium, called after Leukemibacteriumzucineum, it is in China's typical culture collection center preservation, and preserving number is: CCTCC M 205004, preservation date is: on January 7th, 2005.
Bacterial classification source: described rod-shaped bacterium Leukemibacterium zucineum, live in people's the tumor cell line K562 cell, the method of isolating Leukemibacterium zucineum is: people's tumor cell line K562 cell (being provided by the Zhejiang University institute of oncology) is cultivated in the RPMI-1640 complete culture solution, culture temperature is 37 ℃, the methanol extract that adds tobacco leaf, cultivate after 2-7 days, the intracellular bacterium of K562 (Leukemibacterium zucineum) growth obtains the bacterium Leukemibacterium zucineum that grows.Can increase with common inoculum obtaining bacterium Leukemibacterium zucineum, nutrient solution can be common Lb, nutrient broth again, solid medium can be Colombia's blood agar plate, and the MacConkey agar plate is chocolate dull and stereotyped, the lokav flat board, the MH agar plate.Described bacterium Leukemibacterium zucineum being inoculated on the sheep blood agar makes it grow up to colony again.
The growth characteristics of Leukemibacterium zucineum: Leukemibacterium zucineum is a non-zymocyte, poor growth, that 35 ℃ of first culture grew later in 48 hours is tiny, point-like, circle, projection, linen bacterium colony, haemolysis not, and bacterium colony is long to diameter 2-3mm after 72-96 hour, surface drying also is unified into a slice, be wrinkle shape, whole bacterium colony can promote, in the liquid medium within, this bacterium grows at fluid surface, diffusion, the linen mycoderm of formation one deck.Can grow at Mai Kangkai flat board (MacConkey Agar), but on the SS flat board, can not grow.Best with 37 ℃ of growths, in people's cell, can survive.
The form of Leukemibacterium zucineum: be Gram-negative, short and small bacillus.Go smear after long-time the placement again, it is irregular that ne ar becomes, and short and smallly all exists with elongated bacillus.In the brain heart infusion liquid nutrient medium, the bacterium gram's staining is chain.Diameter is 0.5-1.0um, and long is 1.5-3um.Flagella staining is single flagellum, and flagellum is positioned at an end of thalline.Form of bacterium such as rod-short under electron microscope.
The detailed biochemical characteristic of Leukemibacterium zucineum is referring to table 2.
5’-AGAGTTTGATCCTGGCTAGAGCGAACGCTGGCGGCAGGCCTAATACATGCAAGTCGAGCG
GCCCTTCGGGGCAGCGGCGGACGGGTGAGTAACGCGTGGGAATGTGCCCTTTGGTACGGAAC
AACACAGGGAAACTTGTGCTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATCGCCATTGG
AGCAGCCCGCGTTGGATTAGGTAGTTGGTGAGGTAAAGGCTCACCAAGCCGACGATCCATAG
CTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG
CAGCAGTAGGGAATCTTGCGCAATGGGCGAAAGCCTGACGCAGCCATGCCGCGTGAATGATG
AAGGTCTTAGGATTGTAAAATTCTTTCACCGGGGAAGATAATGACGGTACCCGGAGAAGAAG
TCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATT
ACTGGGCGTAAAGGGCGCGTAGGCGGATGTTTAAGTCGGGGGTGAAAGCCCGGGGCTCAACC
TCGGAATTGCCTTCGATACTGGACATCTTGATACGGGAGAGGTGAGTGGAACTCCGAGTGTA
GAGGTGAAATTCGTAGATATTCGGAAGAACACCAGTGGCGAAGGCGACTCACTGGCCCGTTA
CTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCC
GTAAACGATGCGTGCTAGTTGTCGGCATGCATGCATGTCGGTGACGCAGCTAACGCATTAAGC
ACTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAGGGAATTGACGGGGGCCCGCACA
AGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCACCTTTTGACATGCC
CTGATCGCTGGAGAGATCCAGTTTTCCCTTCGGGGACAGGGACACAGGTGCTGCATGGCTGTC
GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCATTAGTT
GCCATCATTAAGTTGGGCACTCTAATGGGACCGCCGGTGGTAAGCCGGAGGAAGGTGGGGAT
GACGTCAAGTCCTCATGGCCCTTACGGGGTGGGCTACACACGTGCTACAATGGCGACTACAG
AGGGTTGCGATGCTGCGAAGCGGAGCTAATCCCTAAAAGTCGTCTCAGTTCGGATTGCACTCT
GCAACTCGAGTGCATGAAGTCGGAATCGCTAGTAGTCGCGGATCAGCATGCCGCGGTGAATA
CGTTCCCGGGCCTTGTACTCACCGCCCGTCGCACCATGGGAGTTGGCTTTACCCGAAGGCGGT
GCGCTAACCAGCAATGGAGGCAGCCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTAA
CAAGGTAGCCGT-3’
Another object of the present invention is because this rod-shaped bacterium Leukemibacteriumzucineum that provides uses in preparation treatment tumour related drugs, and this rod-shaped bacterium the experiment proved that and can kill kinds of tumor cells, but inducing apoptosis of tumour cell.
This rod-shaped bacterium provided by the invention also can be used in preparation contains the biological products of described rod-shaped bacterium Leukemibacterium zucineum, and biological products comprise reagent or test kit.
The material in also available described rod-shaped bacterium Leukemibacterium zucineum and source thereof comprises its genetic material, protein, or other compositions of this bacterium, or use by cultivating the composition that this bacterium obtains, be used to prepare biological products.
Because Leukemibacterium zucineum can enter in the eukaryotic cell (as people's cell), and can be in cell long-term survival, so this bacterium can be used as the compound (as the compound of genetic material, protein, medicine and other any kinds) that carrier carries any kind and enters cell.This purposes is with a wide range of applications on biomedical boundary.
The present invention has done the microbiology evaluation to the novel bacteria that provides, comprise that form, biochemistry, growth, 16SrRNA sequence etc. have all done the detailed experiments evaluation, having disclosed this bacterium is a kind of new bacterium, the bacterium that does not belong to any one known genera is therefore with this bacterium called after Leukemibacterium (generic name) zucineum (planting name).This patent has also disclosed the purposes that Leukemibacterium zucineum has treatment tumour etc.
Description of drawings
Fig. 1: Leukemibacterium zucineum gramstaining result is gram negative bacillus;
Fig. 2: Leukemibacterium zucineum transmission electron microscope photo is shown as bacillus;
Fig. 3: Leukemibacterium zucineum transmission electron microscope photo is shown as bacillus, and paired phenomenon is arranged;
Fig. 4: Leukemibacterium zucineum transmission electron microscope photo, the fine structure of showed cell wall;
Fig. 5: Leukemibacterium zucineum stereoscan photograph is bacillus;
Fig. 6: Leukemibacterium zucineum negative staining Electronic Speculum has a butt flagellum;
Fig. 7: Leukemibacterium zucineum bacterium colony on the blood agar, the dry gauffer shape of bacterium colony gray on the blood agar;
Fig. 8: K562 cell+0.5% (V/V) tobacco leaf methanol extract liquid transmission electron microscope photo after 8 hours, a lot of cavitys are arranged in the cell, mainly be present in the kytoplasm, find L-type bacterium in the cavity, the bacterium size, come in every shape, can find several to dozens of L-type bacteriums in the cell, most of cell film is complete;
Fig. 9: K562 cell+0.5% (V/V) tobacco leaf methanol extract liquid transmission electron microscope photo after 24 hours, cell are all broken, and general form disappears, and sees it almost all being L-type bacterium, and the bacterium size comes in every shape;
Figure 10: the laser co-focusing Photomicrograph, finding has complete Leukemibacteriumzucineum (cell redness, bacterium green) in the K562 cell;
Figure 11: the laser co-focusing Photomicrograph, finding has complete Leukemibacteriumzucineum (cell redness, bacterium green) in Ku8 12 cells;
Figure 12-a: transmission electron microscope shows L-type bacterium in the K562 cell;
Figure 12-b: for amplifying at Figure 12-a square frame place;
Figure 13-a: transmission electron microscope shows L-type bacterium in the Ku812 cell;
Figure 13-b: for amplifying at Figure 13-a square frame place;
Figure 14: the phylogenetic tree of Leukemibacterium zucineum;
Figure 15: Balb/c small white mouse growth curve chart, each, control group and treatment group body weight did not have tangible difference in period;
Figure 16-a: control group heart HE dyeing;
Figure 16-b: experimental group heart HE dyeing;
Figure 17-a:: control group liver HE dyeing;
Figure 17-b: experimental group liver HE dyeing;
Figure 18-a: control group spleen HE dyeing;
Figure 18-b: experimental group spleen HE dyeing;
Figure 19-a: control group lung HE dyeing;
Figure 19-b: experimental group lung HE dyeing;
Figure 20-a: control group kidney HE dyeing;
Figure 20-b: experimental group kidney HE dyeing;
Figure 21-a: control group brain HE dyeing;
Figure 21-b: experimental group brain HE dyeing;
Figure 22-a: control group uterus HE dyeing;
Figure 22-b: experimental group uterus HE dyeing;
Figure 23-a: control group ovary HE dyeing;
Figure 23-b: experimental group ovary HE dyeing;
Figure 24-a: control group stomach HE dyeing;
Figure 24-b: experimental group stomach HE dyeing;
Figure 25-a: control group small intestine HE dyeing;
Figure 25-b: experimental group small intestine HE dyeing;
Figure 26-a: control group large intestine HE dyeing;
Figure 26-b: experimental group large intestine HE dyeing;
Figure 27-a: control group peripheral blood HE dyeing;
Figure 27-b: experimental group peripheral blood HE dyeing;
Figure 28-a: control group marrow HE dyeing;
Figure 28-b: experimental group marrow HE dyeing;
Figure 29-a is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to Ho8910 clone;
Figure 29-b is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to A549 clone;
Figure 29-c is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to D6 clone;
Figure 29-d is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to KBV200 clone;
Figure 29-e is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to Bcap37 clone;
Figure 29-f is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to H9 clone;
Figure 29-g is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to Jurkat clone;
Figure 29-h is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to HL60 clone;
Figure 29-i is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to HL60/ADR clone;
Figure 29-j is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to LS174T clone;
Figure 29-k is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to SW480 clone;
Figure 29-l is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to Pancl clone;
Figure 29-m is the growth-inhibiting graphic representation of the Leukemibacterium zucineum of different concns to RKO clone;
Figure 29-n is that Leukemibacterium zucineum after the hot deactivation is to the inhibition graphic representation of Jurkat clone;
Figure 29-o is that Leukemibacterium zucineum after the hot deactivation is to the inhibition graphic representation of RKO clone;
Figure 29-p is that Leukemibacterium zucineum after the hot deactivation is to the inhibition graphic representation of Ho8910 clone;
Figure 30 is the growth-inhibiting graphic representation of the elutriant of Leukemibacterium zucineum to Jurkat clone;
Figure 31 be Jurkat clone with and handle 24,48,72 hours through Leukemibacterium zucineum after, the variation diagram of G2/M phase cell content;
Figure 32 be Jurkat clone with and handle 24,48,72 hours through Leukemibacterium zucineum after, the variation diagram of inferior 2 times of somatocyte content;
(original magnification is the apoptotic body that Figure 33 occurs through Leukemibacterium zucineum processing back for Jurkat clone: * 400);
Figure 34 handles the DNA ladder electrophorogram that produces after the Jurkat clone for the Leukemibacterium zucineum of different concns;
Figure 35-a is that the detected Jurkat clone of TUNEL test kit was cultivated after 72 hours, the apoptotic FCM figure of Jurkat;
Figure 35-b is that the detected Jurkat clone of TUNEL test kit is through 10
5/ mlLeukemibacterium zucineum handled after 72 hours, the apoptotic FCM figure of Jurkat;
Figure 35-c is that the detected Jurkat clone of TUNEL test kit is through 10
6/ mlLeukemibacterium zucineum handled after 72 hours, the apoptotic FCM figure of Jurkat;
Figure 35-d is that the detected Jurkat clone of TUNEL test kit is through 10
7/ mlLeukemibacterium zucineum handled after 72 hours, the apoptotic FCM figure of Jurkat;
Figure 36 is for handling the variation diagram of the EAC ascites volume in the ICR mouse of back through Leukemibacterium zucineum;
Figure 37 is for handling the variation diagram of the EAC ascites cells sum in the ICR mouse of back through Leukemibacterium zucineum;
Figure 38 is for handling the variation diagram of the HepA ascites volume in the ICR mouse of back through Leukemibacterium zucineum;
Figure 39 is a variation diagram of handling the HepA ascites cells sum in the ICR mouse of back through Leukemibacterium zucineum;
Embodiment
The present invention is described further below in conjunction with specific embodiment, but method related in the scheme and technical parameter can not be interpreted as limitation of the present invention.
The separation of embodiment 1 bacterium
1. experiment material
K562 clone, tobacco leaf powder, methyl alcohol, 1640 cell culture mediums, Colombia's agar basis blood agar (5% sheep blood).
2. experimental technique
Preparation tobacco leaf methanol extract liquid: 20 gram tobacco leaf powder are immersed in the methyl alcohol of 100ml 100%, and placed 7 days in the darkroom, centrifugal 20 minutes of 12000rpm/min, and supernatant is the used tobacco leaf methanol extract liquid of experiment.
The K562 cell inoculation when cell covering in bottom surface surpasses 60%, adds the tobacco leaf methanol extract liquid in 96 orifice plates, and making its final concentration is 0.5%-1% (V/V), 37 ℃ of 5%CO
2Continue to cultivate 24-96 hour, in 96 orifice plates, bacterial growth is arranged.Get the interior bacterial suspension of above-mentioned 96 orifice plates and be inoculated in Colombia's agar basis blood agar (5% sheep blood), 37 ℃, 5%CO
2Cultivated 48-72 hour, and chose bacterium colony and identify.
The source of bacterium is determined in various contrasts:
Divide five groups: 1.K562 cell+substratum+0.5%-1% (V/V) tobacco leaf methanol extract
2.K562 cell+substratum+0.5%-1% (V/V) methyl alcohol
3. substratum+0.5%-1% (V/V) methyl alcohol
4. substratum+0.5%-1% (V/V) tobacco leaf methanol extract
5.K562 cell+substratum.
Each organizes 37 ℃ of 5%CO
2Cultivated 24-96 hour.
3. experimental result
Be inoculated in the K562 cell of 96 orifice plates, behind the tobacco leaf methanol extract of adding 0.5-1%, after 2-14 days bacterial growth arranged, bacterial suspension inoculation is in Colombia's agar basis blood agar (5% sheep blood), 37 ℃, 5%CO
2Cultivated 48-72 hour, colony growth is arranged.Determine to have only first group bacterial growth is just arranged in the experiment of bacterial origin in contrast, other groups do not have bacterial growth (table 1), illustrate that this bacterium comes from the K562 cell interior, and we are its called after Leukemibacterium zucineum.
Table 1: determine Leukemibacterium zucineum source table
| Experimental group | |||||
| ????1 | ??2 | ??3 | ??4 | ??5 | |
| The K562 cell | ????+ | ??+ | ??- | ??- | ??+ |
| Substratum | ????+ | ??+ | ??+ | ??+ | ??+ |
| Methyl alcohol | ????- | ??+ | ??+ | ??- | ??- |
| The tobacco leaf methanol extract | ????+ | ??- | ??- | ??+ | ??- |
| The bacterial growth situation | ????+ | ??- | ??- | ??- | ??- |
This experimental results show that tobacco extract is the essential factor of Leukemibacterium zucineum growth in the irritation cell, if there is not tobacco extract, just can not make the Leukemibacteriumzucineum growth in the K562 cell, can not be separated to Leukemibacterium zucineum, therefore, this experiment provides the method for separating Leukemibacterium zucineum.
The morphology of embodiment 2 Leukemibacterium zucineum is identified
1. experiment material
Colombia's agar basis blood agar (5% sheep blood), the LB liquid nutrient medium, gramstaining reagent, spore staining reagent, OLYMPUS LH50A inverted phase contrast microscope, the JEM-1200 transmission electron microscope (JEOL, Tokyo, Japan), Sterescan 260 flying-spot microscope (Cambridge, England), and the TECNAI10 transmission electron microscope (PHILIPS, Holland).
2. experimental technique
The bacterium that is separated to does conventional gramstaining and spore staining;
The thermo-responsive experiment of the bacterium that is separated to: bacterium 80 ℃, 90 ℃, 100 ℃, behind each 10min and the 20min heat inactivation, is inoculated among the LB and cultivates through 70 ℃.
Leukemibacterium zucineum is inoculated in Colombia's agar basis blood agar, 37 ℃ of 5%CO
2Cultivated 48-72 hour, and observed the bacterium colony characteristics.
Inverted phase contrast microscope is observed the moving situation of Leukemibacterium zucineum among the LB.
After the 1% uranyl acetate solution negative staining, bacterial suspension directly drips on the copper mesh, the transmission electron microscope observing bacterial flagellum.
Scanning electron microscope: Leukemibacterium zucineum fixedly spends the night with 2.5% pentanedial liquid, and then washes 3 times, fixing 3h behind the 1.5% osmic acid liquid, normal developing, dehydration, displacement, CO
2Critical point drying, the gold-plated film of ion sputtering, Sterescan 260 type sem observations.
Transmission electron microscope: Leukemibacterium zucineum is 2.5% preceding the fixing of glutaraldehyde earlier, again with fixing behind 1% the osmic acid, and gradient ethanol dehydration, Epon 812 embeddings, system ultrathin section(ing), uranium nitrate and lead citrate dyeing, TECNAI 10 transmission electron microscope observings.
3. experimental result
Leukemibacterium zucineum is a gram negative bacillus, long 0.5-2um, and wide 0.3-0.5um, cell walls has three-decker, and this bacterium has single flagellum, can be movable in the meat soup liquid nutrient medium.See Fig. 1-6.
Leukemibacterium zucineum is nonspore-bearing negative bacillus, and to thermo-responsive, 70 ℃ of heating 10min get final product killing bacteria.
The dry gauffer shape of Leukemibacterium zucineum bacterium colony gray on blood agar.See Fig. 7.
The growth biochemical characteristic of embodiment 3 Leukemibacterium zucineum
1. experiment material
Nutrient broth, common LB liquid nutrient medium, the MacConkey agar plate, SS selectivity flat board, chocolate dull and stereotyped, the lokav flat board, the MH agar plate, or the like.
2. experimental technique
The bacterium that is separated to is identified its growth biochemical characteristic with API 20NE and ATB ID32 GN and conventional manual method.API 20NE and ATB ID32 GN use in strict accordance with the test kit description of product, conventional manual method is with reference to (Murray, P.R., E.J.Baro, M.A.Pfaller, F.C.Tenover, and.R.H.Yolken (ed.) .1999.Manual of clinical microbiology, 7th ed.American Society forMicrobiology, Washington, D.C.). except that oxydase and catalase test, all growth biochemical tests are seen the result after cultivating 72 hours.
3. experimental result
Biochemistry and cultural characters are:
Table 2: this experiment is separated to the growth and the biochemical characteristic of negative bacillus
| Experimental project | The result |
| Nitre is also tested | ????+ |
| Indole test | ????- |
| Arginine dihydrolase | ????- |
| Urease | ????(+) |
| The toluylic acid desaminase | ????- |
| Hydrolysis β-grape glucoside enzyme | ????(+) |
| Hydrolysising protease | ????(+) |
| Terminal oxidase | ????+ |
| Catalase | ????+ |
| Lysine decarboxylase | ????- |
| Ornithine decarboxylase | ????- |
| Beta-galactosidase enzymes | ????- |
| The DNA enzyme | ????- |
| Acetyl ammonia enzyme | ????- |
| The test of KIA hydrogen sulfide aerogenesis | ????- |
| Methyl red test | ????- |
| The V-P test | ????- |
| Be grown in | ????- |
| The MacConkey agar plate | ????+ |
| The SS flat board | ????- |
| Kligler iron agar | ????- |
| ????LB+0％NaCl | ????+ |
| ????LB+1-4％NaCl | + (the best is 1%NaCl) |
| ????LB+6％NaCl | ????- |
| ????4℃ | ????- |
| ????25℃ | ????+ |
| ????37℃ | ????+ |
| ????47℃ | ????+ |
| The oxidative fermentation test | |
| Glucose | ????O |
| The D-wood sugar | ????- |
| Lactose | ????- |
| Sucrose | ????- |
| Maltose | ????O |
| Pectinose | ????- |
| The D-semi-lactosi | ????O |
| Cellobiose | ????- |
| Rhamnosyl | ????- |
| Fructose | ????O |
| N.F,USP MANNITOL | ????- |
| Mountain Lee's alcohol | ????- |
| Whitfield's ointment | ????- |
| The assimilation test: | |
| Glucose | ????+ |
| Pectinose | ????- |
| Seminose | ????- |
| N.F,USP MANNITOL | ????- |
| The N-acetylglucosamine | ????- |
| Maltose | ????- |
| Gluconate | ????- |
| Capric acid | ????- |
| Hexanodioic acid | ????- |
| Oxysuccinic acid | ????- |
| Citric acid | ????- |
| Toluylic acid | ????- |
| Rhamnosyl | ????- |
| Ribose | ????- |
| Inositol | ????- |
| Sucrose | ????- |
| Methylene-succinic acid | ????- |
| Suberate | ????+ |
| Malonate | ????- |
| Acetate | ????- |
| D-lactate | ????- |
| The L-propionic salt | ????- |
| Whitfield's ointment | ????- |
| Melibiose | ????- |
| The L-Fucose | ????- |
| Mountain Lee's alcohol | ????- |
| Propionic salt | ????- |
| Valerate | ????- |
| Histidine | ????- |
| 5-ketone group gluconate | ????- |
| Glycogen | ????- |
| The 4-hydroxy benzoate | ????- |
| 2-ketone group gluconate | ????- |
| The 3-hydroxybutyric acid | ????+ |
| Hydroxy benzoate | ????- |
| The L-Serine | ????- |
| L-Pu propylhomoserin | ????+ |
+ the positive;-feminine gender; (+) is weak positive; The O oxidation;
Leukemibacterium zucineum catalase, oxidase positive, nitre is also tested the positive.It can assimilate and utilizes glucose, suberate, 3-hydroxybutyric acid and L-Pu propylhomoserin; Urease, hydrolysis β-grape glucoside enzyme and hydrolysising protease are weak positive; It can tardy oxidizing glucose, maltose, D-semi-lactosi and fructose.
Leukemibacterium zucineum indole test, the test of KIA hydrogen sulfide aerogenesis, methyl red test, the V-P test is all negative; It does not have arginine dihydrolase, toluylic acid desaminase, lysine decarboxylase, ornithine decarboxylase, beta-galactosidase enzymes DNA enzyme, acetyl ammonia enzyme; This bacterium can not assimilate the following material of utilization: pectinose, seminose, N.F,USP MANNITOL, the N-acetylglucosamine, maltose, gluconate, capric acid, hexanodioic acid, oxysuccinic acid, citric acid, toluylic acid, rhamnosyl, ribose, inositol, methylene-succinic acid, malonate, sucrose, acetate, D-lactate, the L-propionic salt, Whitfield's ointment, melibiose, the L-Fucose, mountain Lee's alcohol, propionic salt, valerate, Histidine, 5-ketone group gluconate, glycogen, the 4-hydroxy benzoate, 2-ketone group gluconate, hydroxy benzoate, the L-Serine;
Leukemibacterium zucineum can not oxidation can not ferment D-wood sugar, lactose, sucrose, pectinose, cellobiose, rhamnosyl, N.F,USP MANNITOL, mountain Lee's alcohol, Whitfield's ointment.
Leukemibacterium zucineum can be at nutrient broth, and the MacConkey agar plate is chocolate dull and stereotyped, the lokav flat board, and MH agar plate or the like is gone up growth.Can not on SS flat board and kligler iron agar, grow; It can be grown containing in the LB liquid nutrient medium of 0%-4%NaCl, and wherein growth is best in the LB liquid nutrient medium of 1%NaCl, can not grow containing in the LB liquid nutrient medium of 6%NaCl; Under 4 ℃ of temperature, Leukemibacterium zucineum does not grow, and under 25 to 47 ℃, it can be grown, and is best with 37 ℃ of growths.
This experiment provides the method for cultivating Leukemibacterium zucineum.
Embodiment 4:16S rDNA extracts order-checking
1. experiment material
Bacterial genomes DNA extraction test kit (dimension is special clean, Hangzhou), the 16S universal primer:
5 '-AGAGTTTGATCCTGGCTCAG-3 ' (upstream)
5 '-ACGGNTACCTTGTTACGACTT-3 ' (downstream) N represents A, T, and C, any among the G.
PCR reaction common agents box, ABI Prism 377 dna sequencing instrument, the pGEM-T-Easy carrier,
Sequencing primer: 5 '-CCCAGGGTTTTCCCAGTCACGAC-3 ' (upstream)
5 '-TCACACAGGAAACAGCTATGAC-3 ' (downstream)
5 '-GCCTTCGATACTGGACATCTTGA-3 ' (upstream)
5 '-AATTAAACCACATGCTCC ACCG-3 ' (downstream)
2. experimental technique
Extract DNA of bacteria with bacterial genomes DNA extraction test kit, the 16S universal primer carries out pcr amplification, the PCR product cloning is to the pGEM-T-Easy carrier, the transformed competence colibacillus cell, through behind the plate screening, the LB amplification transforms successful bacterium, checks order behind the extraction plasmid, because tested segment＞1500bp, at a pair of primer of indoor design to finish whole pulsating order-checking.Sequencing result is compared in the Genebank/EMBL/DDBJ database.
3. experimental result
16S rDNA sequencing result finds that Leukemibacterium zucineum bacterium is a new bacterial classification in NCBI/EMBL/DDBJ and after the 16S rDNA of known bacterium the comparison.Leukemibacteriumzucineum 16S rDNA total order is classified as:
AGAGTTTGATCCTGGCTAGAGCGAACGCTGGCGGCAGGCCTAATACATGCAAGTCGAGCGG
CCCTTCGGGGCAGCGGCGGACGGGTGAGTAACGCGTGGGAATGTGCCCTTTGGTACGGAACAACA
CAGGGAAACTTGTGCTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATCGCCATTGGAGCAGCC
CGCGTTGGATTAGGTAGTTGGTGAGGTAAAGGCTCACCAAGCCGACGATCCATAGCTGGTCTGAG
AGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGA
ATCTTGCGCAATGGGCGAAAGCCTGACGCAGCCATGCCGCGTGAATGATGAAGGTCTTAGGATTG
TAAAATTCTTTCACCGGGGAAGATAATGACGGTACCCGGAGAAGAAGTCCCGGCTAACTTCGTGC
CAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATTACTGGGCGTAAAGGGCGCGTA
GGCGGATGTTTAAGTCGGGGGTGAAAGCCCGGGGCTCAACCTCGGAATTGCCTTCGATACTGGAC
ATCTTGATACGGGAGAGGTGAGTGGAACTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAAG
AACACCAGTGGCGAAGGCGACTCACTGGCCCGTTACTGACGCTGAGGCGCGAAAGCGTGGGGAGC
AAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGCGTGCTAGTTGTCGGCATGCATG
CATGTCGGTGACGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGATTAAAAC
TCAAGGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCA
GAACCTTACCACCTTTTGACATGCCCTGATCGCTGGAGAGATCCAGTTTTCCCTTCGGGGACAGGG
ACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGC
GCAACCCTCGCCATTAGTTGCCATCATTAAGTTGGGCACTCTAATGGGACCGCCGGTGGTAAGCCG
GAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGGTGGGCTACACACGTGCTACAA
TGGCGACTACAGAGGGTTGCGATGCTGCGAAGCGGAGCTAATCCCTAAAAGTCGTCTCAGTTCGG
ATTGCACTCTGCAACTCGAGTGCATGAAGTCGGAATCGCTAGTAGTCGCGGATCAGCATGCCGCG
GTGAATACGTTCCCGGGCCTTGTACTCACCGCCCGTCGCACCATGGGAGTTGGCTTTACCCGAAGG
CGGTGCGCTAACCAGCAATGGAGGCAGCCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTA
ACAAGGTAGCCGT
4. conclusion:
In the Genebank/EMBL/DDBJ database, do not retrieve the sequence of Leukemibacterium zucineum16S rDNA, therefore can think that the 16S rDNA of Leukemibacterium zucineum is the 16S rDNA of a novel bacterial.
Embodiment 5:Leukemibacterium zucineum genomic dna G+C Determination on content
1. experiment material
Bacterial genomes is extracted test kit (dimension is special clean, Hangzhou), 70% perchloric acid, C
18Reversed-phase column (Atlantis
TMDC
18) intestinal bacteria, A, C, G base standard substance.HEWLETT?Series?1100?HPLC?system。
2. experimental technique
In strict accordance with the explanation that bacterial genomes is extracted test kit, extract Leukemibacteriumzucineum and colibacillary genomic dna, and the DNA that is extracted is carried out purity identify.Behind the highly purified genomic dna frost drying, with perchloric acid hydrolysis one hour in boiling water bath, hydrolysate with water dissolution after centrifugal 20 minutes of 13000rpm, supernatant is HPLC upper prop sample.Use A, C, G configuration standard product solution.
Sample repeats 8 times on the standard substance, obtains A, C, the retention time of G and unit peak area suitable base mole number (representing) with coefficient k
The peak integral area of the volumetric molar concentration/standard substance of k=standard substance
Repeat to prepare sample 3 times, each sample repeats sample 5 times, tries to achieve average peak area.Peak area multiply by corresponding coefficient k and goes out A, C, the mol amount of G.Use formula: G+C=(G+C)/(G+C+2A) tries to achieve the bacterial genomes GC content of Leukemibacterium zucineum.
3. experimental result
Colibacillary G+C content is 51.4%.
The G+C content of Leukemiabacterium zucineum is 71.1%.
4. conclusion:
Colibacillary G+C content is 51.4%, report very near (the foreign literature report is (about 51%) with foreign literature, so the G+C content of the Leukemibacterium zucineum that this method is measured is correct, be 71.1%, illustrate that Leukemibacterium zucineum is the novel bacteria of a high GC content.
Embodiment 6 cytozoicus characteristics
1. experiment material
K562, Ku812, HL60 clone, White Rabbit, ImmunoPure (Protein A) IgGPurification Kit (PIERCE Biotechnology, Rockford, USA).TritonX-100, the goat-anti rabbit two of FITC mark anti-(Santa Cruz, USA), the phallotoxins of rhodamine mark (MolecularProbes, USA), ZEISS LSM510 laser confocal microscope, the JEM-1200 transmission electron microscope (JEOL, Tokyo, Japan).
2. experimental technique
Leukemibacterium zucineum boiling water deactivation 30 minutes is through rabbit auricular vein injection 1 * 10
10After two weeks, strengthen once with the same consumption of quadrat method, get rabbit serum, measuring polyclonal antibody with aggregation tires, when tiring high the time, get blood from rabbit hearts, behind the collection serum, in strict accordance with the anti-Leukemibacterium zucineum of ImmunoPure (Protein A) IgG Purification Kit test kit explanation purified rabbit polyclonal antibody.
K562 cell+0.5%-1% (V/V) tobacco leaf methanol extract 8 hours, and after 24 hours, collecting cell is done conventional transmission electron microscope.
K562 cell, Ku812 cell, HL60 cell and Leukemibacterium zucineum mix at 1: 10, and 37 ℃, 5%CO
2Cultivate 4h and receive cell, make the cell fluorescence immunohistochemical methods.Step is as follows:
Cell behind the infection Leukemibacterium zucineum is thrown on the slide glass that scribbles the bad amino acid of poly.
Under the room temperature, 3.7% Paraformaldehyde 96 is fixed 10 minutes.
Fixing back cell was handled 3-5 minute with 0.2%TritonX-100 (being dissolved in pH7.2, among the 0.1M PBS).50mM TBS, pH 7.2 washes cell.
Add the anti-Leukemibacterium zucineum of rabbit polyclonal antibody, the wet box of room temperature was hatched 2 hours.TBS washes unconjugated one off and resists.
The goat-anti rabbit two that adds the FITC mark is anti-, and under the room temperature, in conjunction with half an hour, the TBS flush away is not anti-in conjunction with two in the secretly wet box.
The phallotoxins transfect cell of rhodamine mark is developed a film.
Glycerine damping fluid mounting, ZEISS LSM510 laser confocal microscope are observed down and are taken pictures.
K562 cell, Ku812 cell, HL60 cell and Leukemibacterium zucineum mix at 1: 10, and 37 ℃, 5%CO
2Collecting cell after cultivation was gone down to posterity 30 days is made cell fluorescence immunohistochemical methods and conventional cell transmission electron microscope.
3. experimental result
Rabbit serum process ImmunoPure (Protein A) the IgGPurification Kit purifying that the potency ratio of collecting is higher obtains the anti-Leukemibacterium zucineum of rabbit polyclonal antibody.
K562 cell+0.5%-1% (V/V) tobacco leaf methanol extract 8 hours, collecting cell is done conventional transmission electron microscope and is found, a lot of cavitys are arranged in the cell, mainly be present in the kytoplasm, find L-type bacterium in the cavity, the bacterium size comes in every shape, and can find several to dozens of L-type bacteriums (Fig. 8) in the cell.Most of cell film is complete.
K562 cell+0.5%-1% (V/V) tobacco leaf methanol extract 24 hours, collecting cell are done conventional transmission electron microscope and are found, cell is all broken, and general form disappears, and sees it almost all being L-type bacterium, the bacterium size, and (Fig. 9) comes in every shape.
Ku812 cell, HL60 cell and Leukemibacterium zucineum mix 37 ℃ 5%COs at 1: 10
2Cultivate 4h and receive cell, make the cell fluorescence immunohistochemical methods, can be observed under ZEISS LSM510 laser confocal microscope has Leukemibacterium zucineum in the cell.(Figure 10-11)
Ku812 cell, HL60 cell and Leukemibacterium zucineum mix 37 ℃ 5%COs at 1: 10
2Collecting cell after cultivation was gone down to posterity 30 days, transmission electron microscope find in the cell born of the same parents L-type bacterium is arranged, but can not find the complete bacterium of cell wall.(Figure 12-a, Figure 12-b, Figure 13-a, Figure 13-b).
This description of test: Leukemibacterium zucineum is the bacterium of L-type in cell.
L-type Leukemibacterium zucineum can with cell symbiosis such as K562, Ku812, HL60, be the L-type during born of the same parents' endoparasitism, generally amplification in a large number keeps a low infection rate in the cell in cell.
The tobacco leaf methanol extract can stimulate the amplification of L-type Leukemibacterium zucineum in the born of the same parents, causes cell rupture death until the bacterium amplification is excessive, and the bacterium of increasing in the cell in initial 24 hours greatly part all is the L-type.Returning to the complete type bacterium of cell wall after the amplification on the bacteria culture medium.
This experimental results show that Leukemibacterium zucineum can enter eukaryotic cell, and in cell long-term survival, so this bacterium can be used as the compound (as the compound of genetic material, protein, medicine and other any kinds) that carrier carries any kind and enters cell.This purposes is with a wide range of applications on biomedical boundary.This experiment also provides the research model of Leukemibacterium zucineum and eukaryotic cell parasitism.This experiment also provides the method that detects Leukemibacterium zucineum.
Embodiment 7 division bacterias
1. experiment material: MEGA2.1 software package.
2. experimental technique:
Set up phylogenetic tree according to Neighbor-Joining method and the two-parameter correction model of Kimura, calculate its Bootstrap value, select for use Rhizobium mongolense as outer population by 1000 sub-samplings.
3. experimental result:
The Bootstrap value indicates all that on Figure 14 the diagram scale represents in each hundred Nucleotide a nucleotide subsitution is arranged.The morphological specificity of cumulated volume research bacterium, the growth biochemical characteristic, in endotrophic characteristic, the 16S homology analysis, we arrive the Alphaproteobacteria order to this division bacteria, Caulobacteraceae section, a new genus, new, and called after: Leukemibacteriumzucineum, represent that this is can be endotrophic bacillus, be found in the Zhejiang University institute of oncology and identify.
The experiment of embodiment 8 animal toxicities
1. experiment material:
Female 6 the week age Balb/c small white mouse, Balb/c nude mice, HE staining reagent.
2. experimental technique:
Laboratory animal is divided into four groups, two experimental group: 10 balb/c small white mouses, 10 balb/c nude mices, two contrasts: 10 balb/c small white mouses, 10 balb/c nude mices.
The Leukemibacterium zucineum of LB fresh culture, with physiological saline give a baby a bath on the third day after its birth all over after, use the resuspended adjusting of physiological saline bacterial concentration to 1 * 10 again
10Cfu/ml.
Experimental group repeats 5 times week about through tail vein injection 100ul bacterial suspension.The control group injection was observed 10 months with the physiological saline of dosage.
3. experimental result:
In 10 months, control group and experimental group do not have death, and without any disease symptoms, body weight and control group do not have the difference (Figure 15) of statistical significance.The main histoorgan heart (Figure 16-a, Figure 16-b), liver (Figure 17-a, Figure 17-b), spleen (Figure 18-a, Figure 18-b), lung (Figure 19-a, Figure 19-b), kidney (Figure 20-a, Figure 20-b), brain (Figure 21-a, Figure 21-b), the uterus (Figure 22-a, Figure 22-b), ovary (Figure 23-a, Figure 23-b), stomach (Figure 24-a, Figure 24-b), small intestine (Figure 25-a, Figure 25-b), large intestine (Figure 26-a, Figure 26-b), peripheral blood (Figure 27-a, Figure 27-b), marrow (Figure 28-a, Figure 28-b) finds that control group and treatment group do not have difference, all show the normal cell weave construction after HE dyeing.Illustrate that Leukemibacterium zucineum does not have toxicity to mouse.
The experiment of embodiment 9 drug-resistance of bacteria
1. experiment material:
Colombia's agar basis blood agar (5% sheep blood), Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29213) medicine Quality Control bacterial strain.Medicine: cefepime; Cephazolin; Cefradine; Cefoperazone; Ceftazime; Cefotaxime; Cefmetazole; Cephalofruxin; Peace Qu Nan; Piperacillin; Oxazacillin; Amikacin; Levofloxacin; Ciprofloxacin; Ethylsisomicin; Ticarcillin/clavulanic acid; Piperacillin/tazobactam; Sulperazone; Imipenum; Meropenem; Vancomycin.
2. experimental technique:
With reference to NCCLS trace broth dilution method (National Committee for Clinical LaboratoryStandards. (2003) .Methods for Dilution Antimicrobial Susceptibility Tests forBacteria That Grow Aerobically.Approved Standard M7-A6,6th ed.NCCLS, Wayne, Pa.), each plate inoculated bacteria concentration 1 * 10
5Cfu/ml cultivated 72-96 hour for 35 ℃.
3. experimental result:
Table 3:Leukemibacterium zucineum drug sensitivity tests
Drug sensitive experiment explanation Leukemibacterium zucineum, comprising than higher the MIC value of a lot of medicines: Cephazolin, and cefradine, cefotaxime, cefmetazole, cephalofruxin is pacified Qu Nan, Oxazacillin, amikacin, Ethylsisomicin.This result illustrates that also this bacterium can be used for drug screening.
1. experimental technique
The microbial culture condition:
Leukemibacterium zucineum bacterium is cultivated in the LB nutrient solution that contains 1% (weight/volume) yeast extract, be positioned over 37 ℃, 5%CO
2Environment in leave standstill and cultivated two days, once remove LB solution with the PBS washing, (PBS) is resuspended for phosphoric acid buffer.The mensuration of Leukemibacterium zucineum concentration is 10 according to the bacterial concentration of 0.5 Maxwell unit
8/ ml.It is 10 that PBS adjusts Leukemibacteriumzucineum concentration
9/ ml is standby.
The cultivation of cell:
Human oophoroma cell line Ho8910, human lung carcinoma cell line A549 and D6, human breast cancer cell strain Bcap37, people T lymphoma cell strain H9 and Jurkat, human promyelocytic leukemia cell strain HL60 and HL60/ADR, human colon cancer cell strain LS174T, SW480 and RKO, human pancreas cancer cell strain Pancl all derive from U.S. ATCC (AmericanTyping Culture Collection) company.Human oral cavity epithelial JEG-3 KBV200 is available from hemopathy institute of the Chinese Academy of Medical Sciences.Various suspension cell lines are cultivated in 96 orifice plates, and every hole contains 2 * 10
4Individual cell.The above-mentioned Leukemibacterium zucineum suspension of adjusting concentration is added in 96 orifice plates, and the concentration of treatment that makes Leukemibacterium zucineum is for being respectively 0,10
5, 10
6, 5 * 10
6, 10
7/ ml, every kind of concentration is provided with three multiple holes.And various attached cells are, cultivate in 96 orifice plates, and every hole contains 0.8 * 10
4Individual cell after cell attachment spends the night, adds 0,10 respectively
5, 10
6, 5 * 10
6, 10
7The Leukemibacterium zucineum of/ml concentration, every kind of concentration is provided with three multiple holes.
Choosing people T lymphoma cell strain Jurkat, three clones of human colon cancer cell strain RKO, human oophoroma cell line Ho8910 carries out cell with heat-inactivated Leukemibacterium zucineum and suppresses experiment.The cultural method of clone is the same.Getting the above-mentioned Leukemibacterium zucineum suspension of adjusting concentration puts and boils 20 minutes killing bacterias in the boiling water, Leukemibacterium zucineum solution after this deactivation is added in 96 orifice plates, make the concentration of treatment of deactivation Leukemibacterium zucineum be equivalent to 0,10 of viable bacteria concentration
5, 10
6, 10
7/ ml, every kind of concentration is provided with three multiple holes.
Above cell is all cultivated in RPMI 1640 substratum (GIBCO, the U.S.) that contain 10% foetal calf serum.Above-mentioned cell is placed 37 ℃, 5%CO
2Continue respectively in the cell incubator to cultivate 72 hours.The cell that taking-up is handled well is counted cell number in every hole with flow cytometer.The experiment triplicate.
2. experimental result:
Shown in Figure 29 (a-m), Ho8910, A549, D6, KBV200, Bcap37, H9, Jurkat, HL60, HL60/ADR, LS174T, SW480, Pancl, RKO clone are to the Leukemibacterium zucineum performance growth-inhibiting phenomenon in various degree of various concentration, and its inhibiting rate and being proportionate property of Leukemibacterium zucineum concentration.When the concentration of Leukemibacterium zucineum is 10
7During/ml, Leukemibacterium zucineum reaches more than 80% the inhibiting rate of most of clone.
Be that Leukemibacterium zucineum after the hot deactivation is to the inhibiting rate of Jurkat, RKO, Ho8910 clone shown in Figure 29 (n-p).Experimental result shows that Leukemibacteriumzucineum after the hot deactivation and live body Leukemibacterium zucineum are suitable to the growth-inhibiting ability of Jurkat, RKO, three clones of Ho8910.
3. conclusion:
That lives all can effectively suppress various growth of human tumor cells with heat-inactivated Leukemibacterium zucineum.
The secretory product of embodiment 11 Leukemibacterium zucineum is to the growth-inhibiting of Jurkat clone:
1. experimental technique
Get Leukemibacterium zucineum bacterial suspension coated plate, cultivated two days in 370C, the 5%CO2 environment, with the Leukemibacterium zucineum on an amount of PBS solution collection flat board, the concentration that makes Leukemibacterium zucineum suspension is 10
11/ ml.6000rpm, 5min is centrifugal, and supernatant liquor is collected in the back, and filters with the sterile filters in 0.22 μ m aperture, gets in this supernatant liquor boiling water of part and boils 20 minutes, and-20 ℃ of preservations are standby.
The Jurkat cell cultures is in 96 orifice plates, and every hole contains 1 * 10
4Individual cell.Handle Jurkat clone with the Leukemibacterium zucineum elutriant of above-mentioned two kinds of processing respectively.Its concentration of treatment is equivalent to 2,4,8,16,32 * 10 respectively
8The resulting solution of wash-out among the Leukemibacterium zucineum of/ml.37 ℃, 5%CO
2Cultivated altogether in the cell incubator 72 hours.Mtt assay detects the OD value of 570nm, and MTT is SIGMA (Saint Louis, Missouri, the U.S.) company's product.
2. experimental result
Figure 30 shows that the elutriant of Leukemibacterium zucineum has certain growth-inhibiting effect to the growth of Jurkat clone, and is concentration dependence dependency, and its inhibiting rate can reach 64.98 ± 4.95%.From 30, find out simultaneously, through identical to the growth-inhibiting ability of Jurkat clone and deactivation not of the Leukemibacteriumzucineum elutriant of boiling water heated and boiled after 20 minutes.
3. conclusion
The Leukemibacterium zucineum elutriant of hot deactivation and not deactivation has strong retarding effect to the growth of Jurkat clone.
Embodiment 12 Leukemibacterium zucineum are to the influence in Jurkat cell line cell cycle
1. experimental technique
The Jurkat cell cultures is in 6 orifice plates, and every hole contains 4 * 10
5Individual cell.The above-mentioned Leukemibacterium zucineum suspension of adjusting concentration is added in 6 orifice plates, and the concentration of treatment that makes Leukemibacterium zucineum is for being respectively 0,10
5, 10
6, 10
7/ ml.Cell is placed 37 ℃, 5%CO
2Continue respectively in the cell incubator to cultivate 24,48,72 hours.Centrifugal collecting cell, press propidium iodide (propidium, PI) test kit (Becton-Dickinson, San Jose, CA, the U.S.) the specification sheets pair cell does PI dyeing, with flow cytometer FACScan (Becton Dickinson, Heidelberg Germany) cycle of analysis Jurkat cell, carries out analyzing and processing with CELLQuest software to data.
2. experimental result
In this experiment, as can be seen, Jurkat clone is after cultivating different time sections from the histogram of Figure 31, and the cell cycle changes considerably less, each cycle ratio quite stable of cell.And with 10
7After the Leukemibacterium zucineum of/ml handled Jurkat cell different time, obvious variation took place in its G2/M phase, and the G2/M phase cell content of especially handling Jurkat cell after 24 hours is increased to 43.48 ± 5.84% from 22.01 ± 2.34%.Prolong the treatment time, the per-cent of G2/M phase cell descends, and the hypodiploid cell increases (as Figure 32) simultaneously.Handled 72 hours at Leukemibacterium zucineum, inferior 2 times of somatocyte are increased to 35.82 ± 4.31%.Illustrate that Leukemibacterium zucineum can cause tumour cell to block in the G2/M phase of cell cycle, the cell of retardance becomes inferior 2 times of somatocyte and death (seeing Table 4, Figure 31 and Figure 32) again.
Table 4:10
7After/ml Leukemibacterium zucineum handles Jurkat clone different time, the variation of cell cycle.
| G 0G 1(％) | S(％) | G 2M(％) | ??Hypodiploid(％) | |
| ??control | 58.57±3.09 | 22.37±2.30 | 22.01±2.34 | ??0.91±0.07 |
| 24 hours | 24.66±3.58 | 30.62±4.27 | 43.48±5.84 | ??1.93±0.80 |
| 48 hours | 51.04±4.58 | 19.95±2.65 | 23.92±3.83 | ??5.38±0.91 |
| 72 hours | 38.44±3.79 | 19.31±1.27 | 7.07±1.27 | ??35.82±4.31 |
3. conclusion
Leukemibacterium zucineum can cause that the cell cycle of tumour cell retardance takes place and the kill tumor cell.
Embodiment 13 Leukemibacterium zucineum inducing apoptosis of tumour cell
1. experimental technique
Auspicious-Giemsa staining detects Jurkat apoptosis form: auspicious-Giemsa stain is the product of Beisuo Biological Technology Co., Ltd., Zhuhai.Jurkat clone is through 10
7/ ml Leukemibacteriumzucineum handled after 72 hours, collecting cell, the centrifugal sheet that gets rid of of 1200rpm * 5min, methyl alcohol fixed cell.To the Jurkat cell dyeing, microscopically is observed according to the specification sheets of the auspicious-Giemsa stain of Bei Suo company.
The detection of dna ladder band: 2 * 10
6The Leukemibacterium zucineum mixed culture 24 of individual Jurkat T cell and different concns, 48, after 72 hours, collecting cell, with 200 μ l cell pyrolysis liquids (10mM Tris-HCl[pH 7.6], 10mM EDTA, 50mM NaCl, 1.25%SDS) abundant suspension cell adds Proteinase K (10 μ g/ml) (Sigma company, Saint Louis, Missouri, U.S.) mixing, 56 ℃ of water-baths 1 hour, adding NaCl solution makes its concentration reach 5M, fully behind the mixing 15,000g is centrifugal, adds the raw spirit of two volumes in the supernatant liquor, centrifugal behind the mixing, precipitate with 50 μ l distilled water dissolving DNAs.Dna solution carries out 1% agarose gel analysis.
TUNEL (TdT-mediated dUTP-biotin nick end labeling, the terminal apoptosis method of original position) apoptosis of analysis Jurkat cell: apoptosis test kit (APO-BRDU a Flow cytomety Kit forApoptosis) is Sigma (Saint Louis, Missour, the U.S.) company's product.2 * 10
6The Jurkat cell through the Leukemibacterium zucineum (10 of different concns
5, 10
6, 10
7/ ml) to handle after 72 hours, collecting cell is according to the accent of the operation instructions analysis of cells of the TUNUL test kit rate of dying.
2. experimental result
As shown in figure 33, Jurkat clone is through 10
7/ ml Leukemibacterium zucineum handled after 72 hours, and apoptotic body appears in the Jurkat cell.
The appearance of dna ladder band is apoptotic sign.Show among Figure 34 that along with the increase of Leukemibacteriumzucineum concentration, trend appears increasing progressively in the DNA ladder of Jurkat clone, when Leukemibacterium zucineum concentration is 10
6Just can detect tangible DNA ladder during/ml, Leukemibacterium zucineum concentration is 10
7The amount of small pieces DNA obviously increases during/ml.What Figure 34 represented is that the Leukemibacterium zucineum of different concns handles the DNA ladder that the Jurkat cell occurred after 72 hours.In the figure, M representation DNA Marker, 1 represents negative control, and 2 represent 10
5/ ml Leukemibacterium zucineum handles Jurkat clone and represented 10 in 72 hours, 3
6/ mlLeukemibacterium zucineum handled Jurkat clone 72 hours, and 4 represent 10
7/ mlLeukemibacterium zucineum handled Jurkat clone 72 hours.
The Leukemibacterium zucineum of different concns handled the Jurkat cell after 72 hours, see Table 1 by the detected apoptosis rate of TUNEL test kit, as can be seen from Table 5, the apoptosis rate of Jurkat cell obviously increases along with increasing of Leukemibacterium zucineum concentration, from 10
59.52 ± 1.29% of/mlLeukemibacterium zucineum processing has been increased to 10
788.38 ± 5.04% of/mlLeukemibacterium zucineum processing.
The detected different concns Leukemibacterium of table 5:TUNEL test kit zucineum handles the apoptosis rate after the Jurkat clone.
| Leukemibacterium zucineum concentration (/ml) | Apoptosis rate (%) |
| Control | ????1.39±0.54 |
| Leukemibacterium | ????9.52±1.29 |
| zucineum?10 5 | ????34.53±3.47 |
| Leukemibacterium | ????88.38±5.04 |
| zucineum?10 6 | |
| Leukemibacterium | |
| zucineum?10 7 |
The Leukemibacterium zucineum that Figure 35 demonstrates Jurkat clone process different concns handled after 72 hours, and the TUNEL test kit is handled, and FCM analyzes the apoptosis rate that draws.The Jurkat cell of the negative control group of Figure 35-a, Figure 35-b represents 10
5The Leukemibacterium zucineum of/ml handles 72 hours apoptosis figure of Jurkat cell, and Figure 35-c represents 10
6The Leukemibacteriumzucineum of/ml handles 72 hours apoptosis figure of Jurkat cell, and Figure 35-d represents 10
7The Leukemibacterium zucineum of/ml handles 72 hours apoptosis figure of Jurkat cell.
2. conclusion:
Leukemibacterium zucineum can cause the apoptosis of Jurkat cell.Therefore Leukemibacterium zucineum inhibition tumour cell is the apoptosis by inducing tumor cell.
Embodiment 14 Leukemibacterium zucineum suppress growth of tumor in vivo
1. experimental technique
Get the ICR female mice (Zhejiang Academy of Medical Sciences) of 18-22g.Every group of mouse quantity of handling is 7.It is 4 groups that Leukemibacterium zucineum is divided into EAC ascites inhibition experiment: control group, per injection 10
8(being designated as the T1 group), 10
9Group (being designated as the T2 group), 10
10The individual Leukemibacterium zucineum bacterium group of group (being designated as the T3 group).Every ICR mouse inoculation 4 * 10
6The EAC of cell number (ehrlich ascites tumor) cell was inoculated after 24 hours, every injected in mice Leukemibacterium of treatment group zucineum suspension, control group injecting normal saline.After this, every other day inject Leukemibacterium zucineum once.Ascites is inoculated back 12 days, takes off neck and puts to death whole mouse, takes out the whole ascites in the mouse, counts its ascites volume and EAC ascites cells number.
Get the ICR female mice of 18-22g.Every group of mouse quantity of handling is 7.Every ICR mouse inoculation 4 * 10
6The rat liver cancer cell HepA ascitic type cell of cell number is inoculated after 24 hours every injected in mice 1ml 10 of treatment group
10The Leukemibacterium zucineum suspension of/ml, the physiological saline of control group injection respective amount.After this, each one day injection Leukemibacterium zucineum once.Inoculate back 12 days from ascites, all put to death mouse, take out the whole ascites in the mouse, count its ascites volume and HepA ascites cells number.
2. experimental result
The result of Figure 36 shows that after the Leukemibacteriumzucineum processing of EAC ascites ICR mouse through different concns, ascites volume and the control group of mouse have tangible minimizing.The ascites volume reduces to the 4.12 ± 1.11ml of 4.80 ± 1.71 and T3 group of 10.33 ± 1.44ml, the T2 group of T1 group from 16.44 ± 2.17ml of control group.
The result of Figure 37 shows that after handling through Leukemibacterium zucineum, EAC ascites cells number also has tangible minimizing.Cell count is from (1505.68 ± 52.38) * 10 from control group
6, T1 group (214.89 ± 15.36) * 10
6, T2 group (84.93 ± 36.49) * 10
6, reduce to (75.08 ± 31.92) * 10 of T3 group
6
In Figure 38, as can be seen through 10
10The Leukemibacterium zucineum processing IC R mouse of amount is after 11 days, and HepA ascites volume of ICR mouse and ascites cells number all significantly reduce with the control group ratio.The ascites volume reduces to (11.6 ± 1.15) ml of Leukemibacterium zucineum treatment group from (16.43 ± 1.92) ml of control group, and HepA ascites cells number is then from (2056.07 ± 384.47) * 10 of control group
6Reduce to (1192.63 ± 211.98) * 10 of Leukemibacterium zucineum treatment group
6, two groups are relatively, and there were significant differences for ascites volume and ascites cells number average, as Figure 39.
3. conclusion: Leukemibacterium zucineum can effectively suppress growth of tumor in vivo, has notable antitumor activity.
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use to greatest extent.Therefore, the preferred specific embodiments of front should be understood that only to illustrate, but not limits the scope of the invention by any way.
＜110〉Zhejiang University
＜120〉a kind of new rod-shaped bacterium and application thereof
<160>7
<210>1
<211>20
<212>DNA
＜213〉bacterial classification (bacteria)
<400>1
agagtttgat?cctggctcag?20
<210>2
<211>21
<212>DNA
＜213〉bacterial classification (bacteria)＜400〉2
acggntacct?tgttacgact?t?21
<210>3
<211>23
<212>DNA
＜213〉bacterial classification (bacteria)
<400>3
cccagggttt?tcccagtcac?gac?23
<210>4
<211>22
<212>DNA
＜213〉bacterial classification (bacteria)
<400>4
tcacacagga?aacagctatg?ac?22
<210>5
<211>23
<212>DNA
＜213〉bacterial classification (bacteria)
<400>5
gccttcgata?ctggacatct?tga?23
<210>6
<211>22
<212>DNA
＜213〉bacterial classification (bacteria)
<400>6
aattaaacca?catgctccac?cg?22
<210>7
<211>1443
<212>DNA
＜213〉bacterial classification (bacteria)
<400>7
agagtttgat?cctggctaga?gcgaacgctg?gcggcaggcc?taatacatgc?aagtcgagcg??60
gcccttcggg?gcagcggcgg?acgggtgagt?aacgcgtggg?aatgtgccct?ttggtacgga??120
acaacacagg?gaaacttgtg?ctaataccgt?atgtgccctt?cgggggaaag?atttatcgcc??180
attggagcag?cccgcgttgg?attaggtagt?tggtgaggta?aaggctcacc?aagccgacga??240
tccatagctg?gtctgagagg?atgatcagcc?acactgggac?tgagacacgg?cccagactcc??300
tacgggaggc?agcagtaggg?aatcttgcgc?aatgggcgaa?agcctgacgc?agccatgccg??360
cgtgaatgat?gaaggtctta?ggattgtaaa?attctttcac?cggggaagat?aatgacggta??420
cccggagaag?aagtcccggc?taacttcgtg?ccagcagccg?cggtaatacg?aagggggcta??480
gcgttgctcg?gaattactgg?gcgtaaaggg?cgcgtaggcg?gatgtttaag?tcgggggtga??540
aagcccgggg?ctcaacctcg?gaattgcctt?cgatactgga?catcttgata?cgggagaggt??600
gagtggaact?ccgagtgtag?aggtgaaatt?cgtagatatt?cggaagaaca?ccagtggcga??660
aggcgactca?ctggcccgtt?actgacgctg?aggcgcgaaa?gcgtggggag?caaacaggat??720
tagataccct?ggtagtccac?gccgtaaacg?atgcgtgcta?gttgtcggca?tgcatgcatg??780
tcggtgacgc?agctaacgca?ttaagcactc?cgcctgggga?gtacggtcgc?aagattaaaa??840
ctcaagggaa?ttgacggggg?cccgcacaag?cggtggagca?tgtggtttaa?ttcgaagcaa??900
cgcgcagaac?cttaccacct?tttgacatgc?cctgatcgct?ggagagatcc?agttttccct??960
tcggggacag?ggacacaggt?gctgcatggc?tgtcgtcagc?tcgtgtcgtg?agatgttggg??1020
ttaagtcccg?caacgagcgc?aaccctcgcc?attagttgcc?atcattaagt?tgggcactct??1080
aatgggaccg?ccggtggtaa?gccggaggaa?ggtggggatg?acgtcaagtc?ctcatggccc??1140
ttacggggtg?ggctacacac?gtgctacaat?ggcgactaca?gagggttgcg?atgctgcgaa??1200
gcggagctaa?tccctaaaag?tcgtctcagt?tcggattgca?ctctgcaact?cgagtgcatg??1260
aagtcggaat?cgctagtagt?cgcggatcag?catgccgcgg?tgaatacgtt?cccgggcctt??1320
gtactcaccg?cccgtcgcac?catgggagtt?ggctttaccc?gaaggcggtg?cgctaaccag??1380
caatggaggc?agccgaccac?ggtagggtca?gcgactgggg?tgaagtcgta?acaaggtagc??1440
cgt
Claims (15)
1. new rod-shaped bacterium, called after Leukemibacterium zucineum, in China's typical culture collection center preservation, preserving number is: CCTCC M 205004.
2. the rod-shaped bacterium of Leukemibacterium zucineum by name according to claim 1 is characterized in that: 16S rRNA has the homologous nucleotide sequence more than 1 99% with SEQ ID NO:
5’-AGAGTTTGATCCTGGCTAGAGCGAACGCTGGCGGCAGGCCTAATACATGCAAGTCGAGCGGCC
CTTCGGGGCAGCGGCGGACGGGTGAGTAACGCGTGGGAATGTGCCCTTTGGTACGGAACAACACAGG
GAAACTTGTGCTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATCGCCATTGGAGCAGCCCGCGT
TGGATTAGGTAGTTGGTGAGGTAAAGGCTCACCAAGCCGACGATCCATAGCTGGTCTGAGAGGATGA
TCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTGCGCA
ATGGGCGAAAGCCTGACGCAGCCATGCCGCGTGAATGATGAAGGTCTTAGGATTGTAAAATTCTTTC
ACCGGGGAAGATAATGACGGTACCCGGAGAAGAAGTCCCGGCTAACTTCGTGCCAGCAGCCGCGGTA
ATACGAAGGGGGCTAGCGTTGCTCGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGATGTTTAAGTC
GGGGGTGAAAGCCCGGGGCTCAACCTCGGAATTGCCTTCGATACTGGACATCTTGATACGGGAGAGG
TGAGTGGAACTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAAGAACACCAGTGGCGAAGGCGA
CTCACTGGCCCGTTACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGT
AGTCCACGCCGTAAACGATGCGTGCTAGTTGTCGGCATGCATGCATGTCGGTGACGCAGCTAACGCA
TTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAGGGAATTGACGGGGGCCCGCAC
AAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCACCTTTTGACATGCCCTG
ATCGCTGGAGAGATCCAGTTTTCCCTTCGGGGACAGGGACACAGGTGCTGCATGGCTGTCGTCAGCT
CGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCATTAGTTGCCATCATTAA
GTTGGGCACTCTAATGGGACCGCCGGTGGTAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCAT
GGCCCTTACGGGGTGGGCTACACACGTGCTACAATGGCGACTACAGAGGGTTGCGATGCTGCGAAGC
GGAGCTAATCCCTAAAAGTCGTCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTCGGAA
TCGCTAGTAGTCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACTCACCGCCCGTC
GCACCATGGGAGTTGGCTTTACCCGAAGGCGGTGCGCTAACCAGCAATGGAGGCAGCCGACCACGGT
AGGGTCAGCGACTGGGGTGAAGTCGTAACAAGGTAGCCGT-3’。
3. rod-shaped bacterium according to claim 1 and 2, it is characterized in that: described rod-shaped bacterium Gram-negative, long 0.5-3um, wide 0.3-1.0um, cell walls has three-decker, have single-ended flagellum, in the meat soup liquid nutrient medium, be chain after the dyeing, the dry gauffer shape of bacterium colony gray on blood agar, haemolysis not, catalase, oxidase positive, nitre are also tested the positive, can assimilate and utilize glucose, suberate, 3-hydroxybutyric acid and L-Pu propylhomoserin; Urease, hydrolysis β-grape glucoside enzyme and hydrolysising protease are weak positive, can tardy oxidizing glucose, maltose, D-semi-lactosi and fructose, on the MacConkey agar plate, grow, can not on SS flat board and kligler iron agar, grow, can grow not containing in the LB liquid nutrient medium of NaCl, can grow containing in the LB liquid nutrient medium of 1-4%, but can not grow containing in the LB liquid nutrient medium of 6%NaCl, under 4 ℃ of temperature, do not grow, under 25 ℃ to 47 ℃, can grow, in people's cell, can survive.
4. according to the arbitrary described rod-shaped bacterium of claim 1-3, preparing the application for the treatment of in the tumour goods.
5. according to the arbitrary described rod-shaped bacterium of claim 1-3, the application in the preparation antitumor drug.
6. according to the arbitrary described rod-shaped bacterium of claim 1-3, the application in the preparation biological products.
7. according to the arbitrary described rod-shaped bacterium of claim 1-3, detect application in the diagnostic kit of this bacterium in preparation.
8. according to the arbitrary described rod-shaped bacterium of claim 1-3, the application of this bacterium in setting up research model.
9. the application of this rod-shaped bacterium according to claim 8 in setting up research model, research model includes but not limited to that this bacterium is used for drug screening and cytozoicus.
10. according to the arbitrary described rod-shaped bacterium of claim 1-3, it is characterized in that:, or use by cultivating the application of composition in the preparation biological products that this rod-shaped bacterium obtains by the material or the composition in its source.
11. rod-shaped bacterium according to claim 10 is characterized in that; The material in source comprises the compound of genetic material, protein, any kind and the various forms of mixtures of these compound formation.
12. rod-shaped bacterium according to claim 10 is characterized in that; The genetic material in source comprises genomic deoxyribonucleic acid, non-genomic group DNA, Yeast Nucleic Acid and any type of nucleic acid material and nucleotide sequence and gene product thereof.
13., it is characterized in that: enter the application in the eukaryotic cell carrying genetic material, protein, compound as carrier according to the arbitrary described rod-shaped bacterium of claim 1-11.
14. one kind is separated from cell as the arbitrary described bacillar method of claim 1-3, it is characterized in that: in cell cultures, add tobacco extract, culture temperature is 37 ℃, cultivate and after 2-14 days bacterial growth is arranged, bacterial suspension inoculation is in Colombia's agar blood agar, cultivated 2-7 days, and obtained bacterium colony.
15. cultivation such as the described bacillar method of 1-3, it is characterized in that: liquid nutrient medium can be common Lb, nutrient broth, solid medium can be Colombia's blood agar plate, and the MacConkey agar plate is chocolate dull and stereotyped, the lokav flat board, the MH agar plate.
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| CN100339474C (en) * | 2005-01-26 | 2007-09-26 | 浙江大学 | Novel bacillus and its separation method and application |
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