CN1174238A - Biomycin producing method utilizing gene engineering technology - Google Patents

Biomycin producing method utilizing gene engineering technology Download PDF

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CN1174238A
CN1174238A CN 97104440 CN97104440A CN1174238A CN 1174238 A CN1174238 A CN 1174238A CN 97104440 CN97104440 CN 97104440 CN 97104440 A CN97104440 A CN 97104440A CN 1174238 A CN1174238 A CN 1174238A
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shengjimycin
spiramycin
isovaleryl
acetylspiramycin
transferase gene
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CN1058295C (en
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王以光
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Shenyang Tonglian Group Co Ltd
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Beijing Capital Science And Technology Group Co ltd
Institute of Medicinal Biotechnology of CAMS
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Abstract

Carbomycin ogenes 4" -isovaleryl transferase is first cloned to spiramycin ogenes to express; the obtained cloned stain is then cultured in inclined soyabean cake powder starch substratum; and through fermentaed liquid extraction, separation and purification, biotechmycin is finally obtained. The said technology has less environment pollution and low expense, and is suitable for industrial production. The biotechmycin is the combination of 4" -acylated spiramycins with 4" -isovaleryl spiramycins II and III as main components. It has powerful antibacterial activity to Gram's positive bacteria and it has low toxicity and cure effect superior to erythromycin, medemycin ahd acetylspiramycin.

Description

A kind of method of utilizing genetic engineering technique MT mycin
The present invention relates to the manufacture method that genetic engineering technique obtains shengjimycin.Shengjimycin be a kind of be the acidylated spiramycin of principal constituent with the isovaleryl Spiramycin Base, to the treatment gram positive bacteria infection effect be better than acetylspiramycin.
Spiramycin Base is because its tissue permeability is good; toxicity is low; it is the microbiotic of a class significant; for further improving its curative effect; they are 4 years old " acidylate of position is main direction; be widely used in clinical acetylspiramycin at present and make by chemical semisynthesis, with chemical process transformation Spiramycin Base except that the shortcoming that environmental pollution is arranged, and acylation reaction azimuth zeroset.Japanese Patent (52-82790) was once reported and transformed Spiramycin Base with microbial process is acidylated spiramycin.The inventor once participated in and utilizes mydecamycin generation bacterium non-activity change strain that Spiramycin Base is carried out the research that microbial transformation obtains propionylspiramycin, and patent (ZL 87 104409.9).Microbial transformation needs to make Spiramycin Base earlier and transforms, and technology is loaded down with trivial details, and transformation efficiency has certain limitation.The inventor once utilized genetic engineering technique to develop 4 " propionylspiramycin, and patent (ZL 90106025.9).Structure activity relationship studies show that acidylated spiramycin curative effect in vivo and 4 " length of position acyl group is relevant, the carbochain of acyl group is long more, fat-soluble good more, curative effect in vivo is good more.
The objective of the invention is to utilize genetic engineering technique to obtain directly to produce the genetic engineering bacterium of shengjimycin.Through the fermentative production shengjimycin, this method acidylate directional property is good, and technology is simple, and cost is lower, and can avoid the environmental pollution that acetylspiramycin caused of chemical process manufacturing.Preliminary pharmacodynamic study shows that shengjimycin curative effect in animal body is better than acetylspiramycin, might become excellent drug, creates good social and economic benefit.
Content of the present invention is with to put division as follows: one.The structure of shengjimycin genetic engineering bacterium
" the isovaleryl transferase gene p66B (deriving from the C.R.Hutchinson of U.S. Wisconsin university professor); its sequence is published in (Gene; 1989; N.85; p.293-301); transform by DNA, change the spiramycin-producing strain protoplastis over to that utilizes Magnamycin A to produce bacterium (Streptomyces thermotoleransATCC11416) 4.The preparation of protoplastis is carried out as follows: at first spiramycin-producing strain (Str.spiramyceticus) bacterial classification (is derived from Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences culture presevation chamber, be published in microorganism journal (1982, V.22, N.1 p.13-16) inclined-plane, dig piece and be seeded to substratum (sucrose 10.3%, K 2SO 40.025%, MgCL 2.6H 2O 1.012%, glucose 1.0%, tryptone 0.1-0.3%, casamino acids 0.05-0.3%, peptone 0.2%, yeast extract 0.2-0.5%, K 2HPO 40.1-0.7% 10ml/L, Tutofusin tris 0.1-0.3M (pH6-8) 100ml/L, trace element solution (ZnCl 20.04%, FeCl 26H 2O 0.02%, CuCl 2.2H 2O0.001%, MnCl 24H 2O 0.001%, NaB 4O 710H 2O 0.001%) 2ml/L, NaOH0.5-2N 2-10ml/L) in, loading amount is the 30-50ml/250ml triangular flask, cultivates 2-4 days for 27-32 ℃.In the above-mentioned same substratum of 2.5-10% inoculum size transferred species, and the glycine of adding 0.1-1%, under similarity condition, cultivated 16-36 hour, collect thalline, wash thalline 2-3 time with 10.3% sucrose solution again, at lysozyme 0.5-3mg/ml P solution (Tutofusin tris 0.2 5-0.4% pH8.0, CaCl 22H 2O 0.25-0.4%, MgCl 26H 2O 0.1-0.3%, sucrose 10.3%, glucose 1% pH7.0-8.0) in, under the 28-38 ℃ of condition bacteriolyze 15-60 minute, obtain protoplastis." isovaleryl transferase gene p66B transforms protoplastis under the PEG media of 15-50% with 4.Acquisition contains 4, and " clone strain of isovaleryl transferase gene [Streptomyces spiramyceticus pBB6-F21] is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number CGMCCNO.0304.Two. the manufacture method of genetically engineered shengjimycin;
Above-mentioned contained 4 " and the spiramycin-producing strain clone strain of isovaleryl transferase gene (Streptomyces spiramyceticus pBB6-F21] containing on the soybean cake powder of 5-30ug/ml thiostrepton-starch slant medium; cultivated 5-14 days in 25-35 ℃; dig the piece method and be inoculated in seed culture medium (glucose 0.5-2%; starch 2-4%; soybean cake powder 1-3%; NaCL0.4%, CaCO 30.3-0.8%), cultivated 40-80 hour, and went into fermention medium (glucose 1-3%, starch 2-6%, fish meal 1-3%, corn steep liquor 0.5-1.5%, NH for 28 ℃ with 2.5-10% inoculum size kind 4NO 30.6%, KH 2PO 40.01-0.08%, MgSO 40.1%, NaCL 1.0%, CaCO 30.3-0.6%), cultivated 72-120 hour for 26-30 ℃, fermented liquid adds 1% Tai-Ace S 150, filters.After filtrate is transferred pH to 8.5-9.0 with 5-10N NaOH, extract with butylacetate, after the butyl ester extracting solution washes with water, change an amount of 0.5-1.0% phosphate buffered saline buffer at pH2-2.5, and then regulate pH to 8.5-9.0 with 2N NaOH, filter and collect the precipitation that forms, in 50-60 ℃ of drying, promptly obtain the shengjimycin sample behind the water thorough washing.Three. the component of shengjimycin and discriminating
Shengjimycin is one group of 4 " Spiramycin Base of acidylate.Its sample distributes through two-phase, centrifugal thin layer analyse or the separation and purification of vacuum chromatography after, through UV spectrum, infrared spectra, mass spectrum and 1H and 13Analyses such as C nucleus magnetic resonance, differentiate that it mainly is subdivided into 4 " the mould III of isovaleryl spiral, II.4 " molecular weight of isovaleryl spiramycin III is 982, molecular formula C 51H 86N 2O 16, UV: λ Max (EtOH)230nm (Fig. 1), IR (KBr): cm -1: 3502.3,2936.3,1735.7,1383.8,1164.9,1054.0 (Fig. 2).FABMS(m/Z)：983(M+H) +825，740，420，406，229，174，142。 1H NMR (500MHz, CDCl 3) (Fig. 3), data see Table 1. 13C NMR (125MHz, CDCl 3) (Fig. 4), data see Table 2.Based on above-mentioned spectroscopic data and to cosy, DEPT, the anatomizing 4 " structure of isovaleryl spiramycin III seen (formula 1) of HETCOR spectrum.4 " molecular weight of isovaleryl spiramycin II is 968, molecular formula C 50H 84N 2O 16, UV: λ Max (EtOH)230nm (Fig. 5), IR (KBr): cm -1: 3500.4,2936.3,1734.8,1381.8,1164.9,1054.9 (Fig. 6).FABMS (m/Z): 969 (M+H) +811,420,392,229,174,142. 1H NMR (500MHz, CDCl 3) (Fig. 7), data see Table 1. 13C NMR (125MHz, CDCl 3) (Fig. 8), data see Table 2.Based on above-mentioned data with 4 " structure of the comparison isovaleryl spiramycin II of isovaleryl spiramycin III spectroscopic data is seen (formula 1).Four. in the shengjimycin other 4 " discriminating of acidylated spiramycin:
Remove in the shengjimycin and contain the isovaleryl spiramycin III, outside the II component, still contain other 4 components.These 4 components warps and 4 " acetylspiramycin and 4 "-propionylspiramycins HPLC (Shim-pack CLC ODS post, methyl alcohol: 1% KH2PO4 (48: 52) makes moving phase, detect wavelength 232nm, flow velocity 1ml/min) compares contrast, it is 4 that these 4 components are differentiated respectively " acetylspiramycin III; II and 4 " propionylspiramycin III, II.Five. the drug effect of shengjimycin
Experiment such as infecting mouse curative effect shows that shengjimycin not only has stronger anti-microbial activity to leather Lan Shi positive bacteria in antibacterial activity in vitro and the body, and to partly removing from office the Lan Shi negative bacteria and anerobe has certain anti-microbial activity.The results are shown in Table 3,4,5,6,7.Measured the protection effect that the shengjimycin mouse peritoneal infects 4 kinds of bacterium (streptococcus pneumoniae, faecalis, Hemolytic streptococcus, golden Portugal bacterium), experimental result shows that shengjimycin curative effect in animal body is better than erythromycin, mydecamycin and acetylspiramycin.The results are shown in Table 8,9.Shengjimycin the results are shown in Table 10,11 in rat tissue's concentration distribution.
Table 14 be " isovaleryl spiramycin III (1) and II's (2) 1HNMR data (500MHz, CDCl 3)
????H ????????????????????????????????1 ????????2
?δ，ppm；J，Hz ??COSY?correlated?H ????δ，ppm；J，Hz
??2ax ??2eq ??3 ??4 ??5 ??6 ??7ax ??7eq ??8 ??9 ??10 ??11 ??12 ??13 ??14ax ??14eq ??15 CH3-16 ??17a ??17b ??18 CH3-19 ??21a ??21b CH3-22 ??23 ?2.25，m ?2.72，dd，J＝11.0，13.3 ?5.14，bd，J＝10.8 ?3.22，bd，J＝9.1 ?3.03，bd，J＝9.1 ?2.15，m ?0.97，m ?1.45，m ?1.92，m ?3，96，dd，J＝3.9，9.6 ?5.61，dd，J＝9.6，15.2 ?6.56，dd，J＝10.5，15.2 ?6.05，dd，J＝10.5，14.7 ?5.73，ddd，J＝3.6，11.3， ?14.7 ?2.12，m ?2.47，m ?5.01，m ?1.24，d，J＝6.1 ?2.30，m ?2.79，dd，J＝11.1，18.2 ?9.64，s ?0.97，d，J＝6.5 ?2.48(not?resolved) ?2.58，dq，J＝7.5，15.7 ?1.20，t，J＝7.5 ?3.51，s(OCH3) ??2eq，3 ??2ax，3 ??2ax，2eq，4 ??3，5 ??4 ??7eq ??7eq，6，8 ??7ax，6，8 ??7ax，CH3-19 ??10 ??9，11 ??10，12 ??11，13 ??12，14ax，14eq ??14eq，13，15 ??14ax，13， ??14ax，CH3-16 ??15 ??17eq ??17ax ??8 ??21b，22 ??21a，22 ??21a，21b ??2.26，bd，J＝13.2 ??2.73，dd，J＝11.0，13.2 ??5.13，bd，J＝11.0 ??3.23，bd，J＝9.1 ??3.87，bd，J＝9.1 ??2.15，m ??0.97，m ??1.45，m ??1.93，m ??3.93，dd，J＝4.0，9.7 ??5.62，dd，J＝9.7，15.2 ??6.55，dd，J＝10.5，15.2 ??6.06，dd，J＝10.5，15.0 ??5.72，ddd，J＝3.6，11.2 ????15.0 ??2.12，m ??2.47，m ??5.06，m ??1.25，d，J＝5.5 ??2.32，dd，J＝3.0，18.3 ??2.82，bdd，J＝11.1，18. ??9.65，s ??0.98，d，J＝6.6 ??2.27，s(CH3CO) ??3.52，s(OCH3)
Table 14 be " isovaleryl spiramycin III (1) and II's (2) 1HNMR data (500MHz, CDCl 3) (continuing)
??H ?????????????????????????????1 ????????2
????δ，ppm；J，Hz ????COSY?correlated?H ????δ，ppm；J，Hz
?1′ ??2′ ??3′ ??4′ ??5′ ??6′ ??7′8′ ??1″ ??2″ax ?2″eq ?4″ ??5″ ??CH3-6″ ??CH3-7″ ??9″ ??10″ ??CH3-11 ?1 ?2ax ?2eq ?3ax ?3eq ?4 ?5 ?CH3-6 ?78 ?4.40，d，J＝7.8 ?3.49，m(overlapped) ?2.45，m(overlapped) ?3.25，m ?3.27，m ?1.19，d，J＝6.1 ?2.50，s[N(CH3)2] ?5.05，d，J＝3.5 ?1.82，dd，J＝4.0，14.1 ?1.99，bd，J＝14.1 ?4.61，d，J＝10.2 ?4.44，dq，J＝6.1，10.2 ?1.12，d，J＝6.1 ?1.10，s ?2.29，d，J＝7.5 ?2.14，m ?″12″ ?0.96，d，J＝6.6 ?4.42，m(overlapped) ?1.49，m ?1.85，m ?1.42，m ?1.87，m ?2.27，m ?3.40，dq，J＝6.2，9.2 ?1.21，d，J＝6.2 ?2.23，s[N(CH3)2] ??2′ ????1′，3′ ????2′，4′ ????3′ ????6′ ????5′ ????2″ax ??2″eq，1″ ????2″ax， ??5″ ????4″，CH3-6″ ????5″ ????10″ ????9″，CH3-11″，12″ ????10″ ????2ax，2eq ??2cq，1，3 ??2ax，1，3 ??2eq，3eq，4 ??2ax，3ax ??3ax，5 ??4，CH3-6 ??5 ????4.42，d，J＝7.5 ????3.50，dd，J＝7.5，10.4 ????2.46，dd，J＝6.7，10.4 ????3.26，m ????3.28，m ????1.19，d，J＝6.6 ????2.50，s[N(CH3)2] ????5.06，d，J＝3.5 ????1.83，dd，J＝4.0，14.2 ????2.00，bd，J＝14.2 ????4.61，d，J＝10.2 ????4.44，dq，J＝6.2，10.2 ????1.13，d，J＝6.2 ????1.11，s ????2.29，d，J＝7.0 ????2.14，m ????0.97，d，J＝6.6 ????4.39，bd，J＝9.3 ????1.48，m ????1.84，m ????1.43，m ????1.85，m ????2.25，m ????3.40，dq，J＝6.2，9.3 ????1.20，d，J＝6.2 ????2.21，s
Table 24 '-isovaleryl spiramycin III (1) and II (2) 13CNMR data (125MHz, CDCl 3)
?C ??????1 ????2 ????C ?????1 ????2
?δ，ppm(DEPT) ??δ，ppm ??δ，ppm(DEPT) ??δ，ppm
?1 ?2 ?3 ?4 ?5 ?6 ?7 ?8 ?9 ?10 ?11 ?12 ?13 ?14 ?15 ?16 ?17 ?18 ?19 ?20 ?21 ?22 ?23 ?1′ ?2′ ?3′ ?4′ ????169.94????(C) ????37.25?????(CH2) ????69.14?????(CH) ????84.69?????(CH) ????77.71?????(CH) ????28.93?????(CH) ????30.11?????(CH2) ????31.85?????(CH) ????79.78?????(CH) ????126.64????(CH) ????135.29????(CH) ????132.18????(CH) ????131.91????(CH) ????41.03?????(CH2) ????69.14?????(CH) ????20.31?????(CH3) ????42.46?????(CH2) ????201.28????(CH) ????15.36?????(CH3) ????173.85????(C) ????27.64?????(CH2) ????8.94??????(CH3) ????62.39?????(CH3) ????103.88????(CH) ????71.60?????(CH) ????68.73?????(CH) ????75,90?????(CH) ????169.96 ????37.14 ????69.10 ????84.80 ????77.60 ????28.86 ????30.03 ????31.97 ????79.79 ????126.59 ????135.41 ????132.25 ????131.84 ????41.02 ????69.18 ????20.33 ????42.42 ????201.21 ????15.37 ????170.79 ????21.25 ????62.45 ????103.74 ????71.61 ????68.71 ????75.96 ??5′ ????6′ ????7′ ????8′ ????1″ ????2″ ????3″ ????4″ ????5″ ????6″ ????7″ ????8″ ????9″ ????10″ ????11″ ????12″ ????1 ??2 ??3 ??4 ??5 ??6 ??7 ??8 ????72.97??(CH) ????19.00??(CH3) ????41.92??(CH3) ????41.92??(CH3) ????97.02??(CH) ????41.67??(CH2) ????69.33??(C) ????77.00??(CH) ????63.48??(CH) ????17.81??(CH3) ????25.32??(CH3) ????172.92?(C) ????43.28??(CH2) ????25.52??(CH) ????22.36??(CH3)* ????22.42??(CH3)* ????100.09?(CH) ????31.17??(CH2) ????18.55??(CH2) ????64.85??(CH) ????73.61??(CH) ????18.79??(CH3) ????40.64??(CH3) ????40.64??(CH3) ????72.95 ????18.98 ????41.92 ????41.92 ????97.04 ????41.69 ????69.34 ????77.00 ????63.50 ????17.81 ????25.34 ????172.92 ????43.29 ????25.52 ????22.36* ????22.41* ????100.29 ????31.21 ????18.47 ????64.84 ????73.75 ????18.79 ????40.66 ????40.66
*Data?in?the?same?culumn?are?interchangeable
The antimicrobial spectrum of table 3 shengjimycin
Bacterial classification Minimum inhibitory concentration MIC μ g/ml
Shengjimycin Acetylspiramycin Erythromycin Mydecamycin
Streptococcus pneumoniae 74Streptococcus pneumoniae P210Streptococcus pneumoniae 936Strep A A3Strep A A15Strep A A18Hemolytic streptococcus A10Hemolytic streptococcus 9438Hemolytic streptococcus 9449Faecalis 9426Faecalis 9431Faecalis 9432Gold Portugal bacterium ATCC25923Gold Portugal bacterium 855Gold Portugal bacterium 858Gold Portugal bacterium 8539Gold Portugal bacterium 8551Form staph 8914Form staph 8932Form staph 8943Form staph 8930Form staph 923Micrococcus catarrhalis 936Micrococcus catarrhalis 939Corynebacterium diphtheriae 921Corynebacterium diphtheriae 922Bacillus typhi murium 931Serratia marcescens 931Serratia marcescens 932Citrobacter 921Citrobacter 922Pathogenic colon bacillus 44155Produce malicious intestinal bacteria 44813Enteroaerogen 951Aerobacter cloacae 925Klebsiella pneumonia 7Proteus mirabilis 49005Shigella dysenteriae 51174Shigella bogdii 51265Salmonella gallinarum 50770 ????0.5 ????0.5 ????0.25 ????0.01 ????0.06 ????0.06 ????0.12 ????0.25 ????0.25 ????0.5 ????0.5 ????0.5 ????0.5 ????0.06 ????0.06 ????0.06 ????0.03 ????1 ????0.5 ????1 ????0.12 ????0.25 ????64 ????0.25 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????8 ????32 ????＞64 ????1 ????1 ????0.5 ????0.06 ????0.06 ????0.06 ????0.25 ????2 ????0.25 ????0.5 ????0.25 ????0.25 ????1 ????4 ????8 ????16 ????2 ????2 ????0.5 ????2 ????0.5 ????2 ????32 ????1 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????16 ????64 ????＞64 ????0.5 ????1 ????0.5 ????0.03 ????0.01 ????0.01 ????0.06 ????0.06 ????0.12 ????1 ????1 ????1 ????0.5 ????4 ????＞128 ????＞128 ????0.06 ????32 ????0.06 ????0.06 ????0.25 ????0.06 ????＞64 ????＞64 ????16 ????32 ????16 ????＞64 ????＞64 ????64 ????＞64 ????8 ????8 ????64 ????64 ????＞64 ????＞64 ????2 ????2 ????16 ????0.5 ????1 ????0.5 ????0.03 ????0.06 ????0.02 ????0.25 ????0.5 ????16 ????2 ????1 ????2 ????0.5 ????0.12 ????＞128 ????0.5 ????0.12 ????1 ????0.06 ????1 ????2 ????1 ????8 ????32 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????64 ????＞64 ????＞64 ????＞64 ????＞64 ????＞64 ????2 ????64 ????＞64
Table 4 shengjimycin is to 22 strain anerobe anti-microbial effects
Bacterial classification ????????????????????????MIC????μg/ml
Shengjimycin Mydecamycin Acetylspiramycin Erythromycin Metronidazole plain BP.98 99
The many types of bacteroid of bacteroides fragilis bacteroides fragilis bacteroides fragilis bacteroides fragilis bacteroides vulgatus bacteroides vulgatus bacteroides distasonis bacteroides distasonisATCC29741Many types of genera bacillus oral cavity Pu Shi bacillus is not understood sugared Pu Shi bacillus dyspepsiacoccus peptostreptococcus clostridium perfringens ATCC15132C.perfringens Propionibacterium Propionibacterium lactobacillus lactobacillus yeast-like fungi yeast-like fungi ????0.5 ????0.5 ????1 ????0.5 ????0.5 ????0.5 ????0.5 ????0.5 ????0.5 ????0.5 ????0.5 ????0.5 ????0.5 ????0.5 ????1 ????1 ????0.5 ????0.5 ????0.5 ????1 ????＞64 ????＞64 ????1 ????1 ????1 ????1 ????2 ????1 ????1 ????1 ????1 ????1 ????1 ????1 ????1 ????1 ????4 ????4 ????1 ????1 ????1 ????1 ????＞64 ????＞64 ????0.5 ????0.5 ????1 ????0.5 ????0.5 ????0.5 ????1 ????0.5 ????0.5 ????0.5 ????0.5 ????0.5 ????0.5 ????0.5 ????1 ????2 ????0.5 ????0.5 ????0.5 ????0.5 ????＞64 ????＞64 ????1 ????0.25 ????1 ????0.25 ????0.25 ????0.25 ????0.25 ????0.25 ????0.25 ????0.25 ????0.25 ????0.25 ????0.25 ????0.5 ????0.5 ????1 ????0.5 ????0.25 ????0.25 ????0.5 ????＞64 ????＞64 ????0.5 ????0.25 ????0.5 ????0.25 ????0.25 ????0.25 ????0.25 ????0.25 ????0.5 ????0.5 ????0.25 ????0.25 ????0.25 ????0.5 ????0.25 ????0.5 ????2 ????1 ????16 ????4 ????＞64 ????＞64
Table 5 shengjimycin and other antibiotic are to G +The bacterium anti-microbial activity relatively
Test organisms and strain number Medicine MIC scope μ g/ml ????MIC 50????μg/ml ????MIC 90????μg/ml
Streptococcus pneumoniae (43) Shengjimycin acetylspiramycin erythromycin mydecamycin ?0.03-64 ?0.03-＞64 ?0.005-＞64 ?0.03-＞64 ????0.12 ????0.12 ????0.01 ????0.12 ????4 ????8 ????64 ????64
Hemolytic streptococcus (55) Shengjimycin acetylspiramycin erythromycin mydecamycin ?0.12-64 ?0.25-＞64 ?0.05-＞64 ?0.002-＞64 ????0.5 ????0.25 ????0.12 ????0.25 ????16 ????32 ????16 ????64
Gold Portugal bacterium (product β-Nei Xiananmei) (86) Shengjimycin acetylspiramycin erythromycin mydecamycin ?0.06-＞64 ?0.5-＞64 ?0.12-＞64 ?0.12-＞64 ????2 ????4 ????64 ????1 ????＞64 ????＞64 ????＞64 ????＞64
Gold Portugal bacterium (not producing β-Nei Xiananmei) (76) Shengjimycin acetylspiramycin erythromycin mydecamycin ?0.03-＞64 ?0.25-＞64 ?0.03-＞64 ?0.03-＞64 ????16 ????4 ????64 ????4 ????＞64 ????＞64 ????＞64 ????＞64
Form staph (product β-Nei Xiananmei) (59) Shengjimycin acetylspiramycin erythromycin mydecamycin ?0.5-＞64 ?0.5-＞64 ?0.03-＞64 ?0.06-＞64 ????32 ????32 ????32 ????32 ????＞64 ????＞64 ????＞64 ????＞64
Form staph (not producing β-Nei Xiananmei) (67) Shengjimycin acetylspiramycin erythromycin mydecamycin ?0.03-＞64 ?0.12-＞64 ?0.03-＞64 ?0.06-＞64 ????64 ????64 ????64 ????32 ????＞64 ????＞64 ????＞64 ????＞64
Table 6 shengjimycin and other antibiotic compare hemophilus influenza and gonococcus anti-microbial activity
Test organisms and strain number Medicine MIC scope μ g/ml ????MIC 50????μg/ml ????MIC 90????μg/ml
Hemophilus influenza (30) Shengjimycin acetylspiramycin erythromycin mydecamycin ??0.03-32 ??0.03-＞64 ??0.03-＞64 ??0.01-＞64 ????0.06 ????0.06 ????0.06 ????0.03 ????4 ????4 ????1 ????2
Gonococcus (10) Shengjimycin acetylspiramycin erythromycin mydecamycin ??0.12-16 ??0.12-64 ??0.12-64 ??0.12-64 ????2 ????4 ????1 ????0.5 ????8 ????8 ????8 ????2
Table 7 shengjimycin is to anti-erythromycin gold Portugal bacterium of clinical separation and streptococcus pneumoniae anti-microbial activity
Test organisms and strain number Medicine MIC scope μ g/ml ????MIC 50????μg/ml ????MIC 90????μg/ml
Anti-erythromycin gold Portugal bacterium (product β-Nei Xiananmei) (20) Shengjimycin acetylspiramycin erythromycin mydecamycin ?0.06＞128 ?1-＞128 ?32-＞128 ?0.12-＞128 ????1 ????8 ????128 ????0.25 ????16 ????128 ????＞128 ????16
Anti-erythromycin gold Portugal bacterium (not producing β-Nei Xiananmei) (13) Shengjimycin acetylspiramycin erythromycin mydecamycin ?0.06-＞128 ?0.25-＞128 ?32-＞128 ?0.5-＞128 ????0.5 ????4 ????64 ????64 ????16 ????128 ????128 ????128
Anti-erythromycin streptococcus pneumoniae (11) Shengjimycin acetylspiramycin erythromycin mydecamycin ??0.5-64 ??1-＞64 ??16-＞64 ??16-＞64 ????2 ????8 ????32 ????32 ????32 ????64 ????＞64 ????64
Table 8 shengjimycin infects 6 strain G+ bacterium protection effect test bacterium challenging dose medicine ED50 (mg/kg) (effective dose) streptococcus pneumoniae 8 7.3 * 10 to mouse peritoneal 4Shengjimycin 12.62
Acetylspiramycin 14.10
Erythromycin 66.48
Mydecamycin 96.19 streptococcus pneumoniaes 18 6.7 * 10 3Shengjimycin 8.03
Acetylspiramycin 11.28
Erythromycin 30.08
Mydecamycin 55.28 Hemolytic streptococcuss 772 7.2 * 10 3Shengjimycin 30.08
Acetylspiramycin 42.07
Erythromycin 91.08
Mydecamycin 119.66 faecalis 27 7.2 * 10 3Shengjimycin 47.36
Acetylspiramycin 51.27
Erythromycin 111.94
Mydecamycin 202.24 faecalis 32 8.9 * 10 4Shengjimycin 124.84
Acetylspiramycin 145.36
Erythromycin 152.85
Mydecamycin 208.69 gold medal Portugal bacterium 32 8.9 * 10 4Shengjimycin 91.08
Acetylspiramycin 158.19
Mydecamycin 253.44
Table 9 shengjimycin therapeutic index Hemolytic streptococcus shengjimycin 76.9 in animal body
Acetylspiramycin 37.1 streptococcus pneumoniae shengjimycins 700.3
Acetylspiramycin 385.5 gold medal Portugal bacterium shengjimycins 37.2
Acetylspiramycin 20.4 obviously is better than acetylspiramycin by the visible shengjimycin of table security and validity in vivo.
Table 10 shengjimycin is at rat tissue's CONCENTRATION DISTRIBUTION (ug/g) tissue 0.5h 1h 1.5h 2.0h 3.0h 4.0h 6.0h 8.0h 12h heart 9.18 11.02 8.40 6.78 9.64 11.57 9.72 5.02 lungs 10.65 10.86 15.17 18.08 8.63 16.26 15.51 6.70 livers 6.14 68.96 69.60 100.52 105.86 102.48 86.94 65.24 44.40 spleens 1.77 17.63 22.54 29.34 40.66 33.58 42.16 39.63 34.44 kidneys 11.28 12.00 11.12 25.44 18.30 42.34 22.92 15.82 stomaches 8.82 16.40 19.19 17.60 11.91 12.20 12.96 10.28 12.02 intestines 85.69 24.44 38.94 17.82 32.59 25.52 31.70 11.08 fat 3.04 5.18 4.12 2.28 1.04 2.72 1.66 1.44 muscle 3.37 1.62 1.00 1.22 2.08 1.92 2.60 1.58 uterus 3.34 blood 2.10 1.46 1.28 0.84 18.20 1.33 0.84 0.47
Table 11 shengjimycin is at rat tissue's CONCENTRATION DISTRIBUTION (ug/g) (continuing) tissue 14h 16h 20h 36h 48h 70h 94h 118h 142h 166h heart 4.31 2.90 2.38 4.12 2.06 1.74 0.64 lungs 8.03 8.46 6.59 5.66 8.32 4.24 2.35 2.47 livers 40.15 30.02 24.48 13.25 17.48 13.85 9.10 3.52 spleens 33.92 32.64 25.81 29.82 25.87 16.64 15.02 5.52 4.46 kidneys 14.66 17.34 13.40 15.41 11.50 10.43 5.66 4.88 stomaches 7.40 6.26 4.75 5.83 5.76 4.08 1.68 1.34 intestines 10.68 8.82 6.42 9.58 6.92 6.70 fat 1.54 1.64 1.47 1.14 muscle 1.09 1.11 0.54 0.38 uterus 5.16 2.42 2.20 2.22 blood 0.46 0.32 0.25 0.27
Pharmacokinetic shows that shengjimycin is more stable in animal body, can keep the longer effective concentration time.Shengjimycin Ames (causing prominent) experimental result is negative.Oral acute toxicity (the LD of the rat of shengjimycin and mouse 50) be＞4000mg/kg.Description of drawings:
Fig. 1. 4 " UV spectrum of isovaleryl spiramycin III
Fig. 2. 4 " infrared spectra of isovaleryl spiramycin III
Fig. 3. 4 " the isovaleryl spiramycin III 1H NMR spectrum
Fig. 4. 4 " the isovaleryl spiramycin III 13C NMR spectrum
Fig. 5. 4 " UV spectrum of isovaleryl spiramycin II
Fig. 6. 4 " infrared spectra of isovaleryl spiramycin II
Fig. 7. 4 " the isovaleryl spiramycin II 1H NMR spectrum
Fig. 8. 4 " the isovaleryl spiramycin II 13C NMR spectrum Formula (1) 4 " isovaleryl spiramycin III
4 " isovaleryl spiramycin II

Claims (4)

1. method of utilizing genetic engineering technique MT mycin, its feature comprises the steps:
A. " the isovaleryl transferase gene clone is to spiramycin-producing strain, makes it to express, " the spiramycin-producing strain clone strain of isovaleryl transferase gene (the Streptomyces spiramyceticus (p66B)-F21) that obtains to contain 4 Magnamycin A to be produced bacterium 4.
B. with (Streptomyces spiramyceticus (p66B)-F21) tiltedly cultivates on the substratum at soybean cake powder-starch, through seed and fermentation culture, from broth extraction, separation, purifying, obtains shengjimycin.
2. a kind of method of utilizing genetic engineering technique MT mycin as claimed in claim 1; it is characterized in that Magnamycin A is produced bacterium 4 that " isovaleryl transferase gene p66B is under 15-50% concentration PEG media; change in the protoplastis of spiramycin-producing strain (Streptomycesspiramyceticus), " the spiramycin-producing strain clone strain of isovaleryl transferase gene (the Streptomyces spiramycelicus (p66B)-F21) that obtains to contain 4.
3. a kind of according to claim 1 method of utilizing genetic engineering technique MT mycin; it is characterized in that to contain 4 that " the spiramycin-producing strain clone strain of isovaleryl transferase gene; containing on the soybean cake powder of 5-30ug/ml thiostrepton-starch slant medium; cultivated 5-14 days in 25-35 ℃; dig the piece method and be inoculated in seed culture medium (glucose 0.5-2%, starch 2-4%, soybean cake powder 1-3%; NaCL 0.4%; CaCO3 0.3-0.8%) was cultivated 40-80 hour for 28 ℃, with 2.5-10% inoculum size kind people fermention medium (glucose 1-3%; starch 2-6%; fish meal 1-3%, corn steep liquor 0.5-1.5%, NH4NO3 0.6%; KH2PO4 0.01-0.08%; MgSO4 0.1%, and NaCL 1.0%, CaCO3 0.3-0.6%); cultivated 72-120 hour for 26-30 ℃, obtain fermented liquid.
4. a kind of according to claim 1 method of utilizing genetic engineering technique MT mycin is characterized in that adding 1% Tai-Ace S 150 in fermented liquid, filter.After filtrate is transferred pH to 8.5-9.0 with 5-10N NaOH, extract with butylacetate, after the butyl ester extracting solution washes with water, change an amount of 0.5-1.0% phosphate buffered saline buffer at pH2-2.5, and then regulate pH to 8.5-9.0 with 2N NaOH, filter and collect the precipitation that forms, in 50-60 ℃ of drying, promptly obtain the shengjimycin sample behind the water thorough washing.
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