Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum.

The Zymomonas mobilis genes for ethanol production have been integrated into the chromosome of Klebsiella oxytoca M5A1. The best of these constructs, strain P2, produced ethanol efficiently from cellobiose in addition to monomeric sugars. Utilization of cellobiose and cellotriose by this strain eliminated the requirement for external beta-glucosidase and reduced the amount of commercial cellulase needed to ferment Solka Floc SW40 (primarily crystalline cellulose). The addition of plasmids encoding endoglucanases from Clostridium thermocellum resulted in the intracellular accumulation of thermostable enzymes as coproducts with ethanol during fermentation. The best of these, strain P2(pCT603T) containing celD, was used to hydrolyze amorphous cellulose to cellobiose and produce ethanol in a two-stage process. Strain P2(pCT603T) was also tested in combination with commercial cellulases. Pretreatment of Solka Floc SW40 at 60 degrees C with endoglucanase D substantially reduced the amount of commercial cellulase required to ferment Solka Floc. The stimulatory effect of the endoglucanase D pretreatment may result from the hydrolysis of amorphous regions, exposing additional sites for attack by fungal cellulases. Since endoglucanase D functions as part of a complex in C. thermocellum, it is possible that this enzyme may complex with fungal enzymes or bind cellulose to produce a more open structure for hydrolysis.     

Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum.

The Zymomonas mobilis genes for ethanol production have been integrated into the chromosome of Klebsiella oxytoca M5A1. The best of these constructs, strain P2, produced ethanol efficiently from cellobiose in addition to monomeric sugars. Utilization of cellobiose and cellotriose by this strain eliminated the requirement for external beta-glucosidase and reduced the amount of commercial cellulase needed to ferment Solka Floc SW40 (primarily crystalline cellulose). The addition of plasmids encoding endoglucanases from Clostridium thermocellum resulted in the intracellular accumulation of thermostable enzymes as coproducts with ethanol during fermentation. The best of these, strain P2(pCT603T) containing celD, was used to hydrolyze amorphous cellulose to cellobiose and produce ethanol in a two-stage process. Strain P2(pCT603T) was also tested in combination with commercial cellulases. Pretreatment of Solka Floc SW40 at 60 degrees C with endoglucanase D substantially reduced the amount of commercial cellulase required to ferment Solka Floc. The stimulatory effect of the endoglucanase D pretreatment may result from the hydrolysis of amorphous regions, exposing additional sites for attack by fungal cellulases. Since endoglucanase D functions as part of a complex in C. thermocellum, it is possible that this enzyme may complex with fungal enzymes or bind cellulose to produce a more open structure for hydrolysis.     

致病疫霉拮抗菌株YR-7 的分离鉴定及其活性物质

【目的】从黄河边的农田土壤中分离筛选拮抗致病疫霉的粘细菌,鉴定目标菌株,分析其发酵上清液的稳定性及对马铃薯晚疫病菌的抑制效果,为活性物质分离鉴定及抗马铃薯晚疫病菌生物农药的研发奠定基础。【方法】采用兔粪诱导法分离菌株,通过平板对峙法筛选对马铃薯晚疫病菌有拮抗作用的粘细菌,通过形态特征、生理生化特征以及16S r RNA基因序列分析对菌株进行鉴定。采用称重法测定菌株生长曲线,通过平皿法测定菌株不同生长时期发酵上清液对致病疫霉的菌丝生长抑制率和浓缩发酵上清液的稳定性。通过马铃薯离体叶片涂布浓缩发酵上清液和接种病原菌孢子悬浮液法,测定该菌株对马铃薯晚疫病的防病作用。【结果】从土壤样品中共分离获得7株粘细菌,其中4株拮抗致病疫霉,拮抗效果最强的为YR-7菌株,菌丝的生长抑制率为96%,该菌株被鉴定为Myxococcus xanthus。培养7 d后,菌株发酵上清液对致病疫霉的抑制活性趋于稳定。浓缩发酵上清液经30-50°C处理后,对致病疫霉菌丝的生长抑制率可达50.90%,高于50°C时抑菌活性逐渐下降,90°C处理后菌丝的生长抑制率仍可达25.45%。浓缩发酵上清液在p H 4.0-9.0条件下比较稳定,保持40.21%以上菌丝的生长抑制率,当p H4.0或p H9.0时,抗菌活性显著降低。活性物质不能被蛋白酶降解,其抗菌活性不受紫外线、自然光照射的影响。对马铃薯离体叶片的生防效果检测表明,YR-7的浓缩发酵上清液处理组叶片相对病斑面积仅为0.35%,对照组的相对病斑面积高达68.19%。【结论】粘细菌菌株YR-7可以产生抗马铃薯晚疫病菌的次生代谢产物,抗菌活性物质具有较好的稳定性,可以有效抑制致病疫霉侵染马铃薯叶片,具有开发成抗马铃薯晚疫病生物农药的潜在价值。     

拟柱孢藻N8全基因组测序及磷吸收转运通路的比较分析

为从分子水平解析热带特征性蓝藻-拉氏拟柱孢藻(Cylindrospermopsis raciborskii,简称拟柱孢藻)环境适应机制,研究以广东省镇海水库中分离的拟柱孢藻藻株N8为材料,采用PacBio单分子实时测序技术(Single Molecule Real-Time, SMRT)进行测序,并初步进行全基因组特征的比较分析。结果显示拟柱孢藻N8全基因组大小为3.857 Mb, GC含量为40.13%,预测编码基因个数为3598个,在COG、KEGG和GO数据库中注释到的基因数分别为2429、1664和2244个。N8藻株与美国国立生物技术信息中心(NCBI)上公布的27株拟柱孢藻基因组大小和GC含量基本一致,但N8的编码基因数最多。基于拟柱孢藻全基因组单拷贝基因的系统进化树表明N8藻株与韩国藻株GIHE 2018的亲缘关系最近。N8藻株的基因组中未发现拟柱孢藻毒素(CYN)和石房蛤毒素(STX)合成酶基因簇,表明该藻株不产蓝藻毒素CYN和STX。对N8藻株和其他7藻株的磷吸收转运通路比较分析表明,这些藻株均拥有较为完整的磷吸收转运基因(双组分调节系统、低亲和力无机磷转运基因、高亲...     

Octene Epoxidation by a Cold-Stable Alkane-Oxidizing Isolate of Pseudomonas oleovorans

The isolation of an alkane-oxidizing strain of Pseudomonas oleovorans which maintains its viability at 5 C is described. This strain epoxidates 1-octene at a rate five times that of the parent strain. The most efficient substrates for induction of the epoxidase are C(7), C(8), and C(9), although C(5) to C(12) also serve as growth substrates and inducers. The greater rate may be attributed to an enhanced general stability of the cells as opposed to a modification of the enzyme system involved.     

Survival of cultured cells in the cold : A kinetic study with special reference to the effect of serum in culture media

The survival at 4 °C of mouse fibroblasts (strain L-929) and rat liver cells (strain JTC-25·P5) was kinetically analysed after they had been pre-incubated at 37 °C in medium with or without supplement of serum. Both the composition of medium used for preincubation at 37 °C and that employed for storage at 4 °C had influence on the survival period.When the cells had been grown at 37 °C in Eagle minimal essential medium (MEM) alone, they rapidly lost their viability at 4 °C from the beginning. However, when grown at 37 °C in MEM supplemented with calf serum, they maintained viability at 4 °C for about 16 days and 8 days for L cells and JTC-25·P5 cells respectively, before the initiation of rapid loss of viability. The presence of macromolecular fraction of calf serum in the medium during preincubation was found to be responsible for the prolongation of survival at 4 °C.     

Distribution of conserved and specific epitopes on the VP8 subunit of rotavirus VP4.

Three cDNA clones comprising the VP8 subunit of the VP4 of human rotavirus strain KU (VP7 serotype G1; VP4 serotype P1A) G1 were constructed. The corresponding encoded peptides were designated according to their locations in the VP8 subunit as A (amino acids 1 to 102), B (amino acids 84 to 180), and C (amino acids 150 to 246 plus amino acids 247 to 251 from VP5). In addition, cDNA clones encoding peptide B of the VP8 subunit of the VP4 gene from human rotavirus strains DS-1 (G2; P1B) and 1076 (G2; P2) were also constructed. These DNA fragments were inserted into plasmid pGEMEX-1 and expressed in Escherichia coli. Western immunoblot analysis using antisera to rotavirus strains KU (P1A), Wa (P1A), DS-1 (P1B), 1076 (P2), and M37 (P2) demonstrated that peptides A and C cross-reacted with heterotypic human rotavirus VP4 antisera, suggesting that these two peptides represent conserved epitopes in the VP8 subunit. In contrast, peptide B appears to be involved in the VP4 serotype and subtype specificities, because it reacted only with the corresponding serotype- and subtype-specific antiserum. Antiserum raised against peptide A, B, or C of strain KU contained a lower level of neutralizing activity than did that induced by the entire VP8 subunit. In addition, the serotype-specific neutralizing activity of anti-KU VP8 serum was ablated after adsorption with the KU VP8 protein but not with a mixture of peptides A, B, and C of strain KU, suggesting that most of the serotype-specific epitopes in the VP8 subunit are conformational and are dependent on the entire amino acid sequence of VP8.     

Pathogenicity of entomopathogenic fungi on hibernating pupae of Cameraria ohridella Deschka & Dimic 1986 (Lepidoptera, Gracillariidae). Part 1: Pathogenicity against the naked pupa

Strains from Paecilomyces fumosoroseus, Lecanicillium muscarium, Metarhizium anisopliae and Beauveria bassiana were examined in standardized Biotest to control the horse-chestnut leaf miner (Cameraria ohridella) in her pupal stage in winter. The fungi were pathogenic against the hibernating pupae of Cameraria ohridella at dose of 1.9 x 10(7) conidia/ml. They were aggressive, led to infection, death and mouldiness of naked pupae. Even at low temperature of 5 degrees C and 12 degrees C. L. muscarium strain V24 showed the highest pathogenicity after 4 weeks against this host, close followed by P. fumosoroseus strain P6. M. anisopliae strain 72 and 8. bassiana strain B412 were also pathogen but after a long-time period. Experiments gave information for general susceptibility of naked pupae of C. ohridella under low temperatures against entomopathogenic fungi. In further examinations it has to be tested, whether fungi can infected, when the pupae stay in their natural surroundings, the pupal cell in the leaf.     

Hyperinduction of nitrile hydratase acting on indole-3-acetonitrile in Agrobacterium tumefaciens

 We investigated the optimum conditions for the formation of nitrile hydratase (NHase), which acts on indole-3-acetonitrile, in Agrobacterium tumefaciens. Good inducers for enzyme formation have been found to be roughly classified into three representative types of amides such as pivalamide, crotonamide and ɛ-caprolactam. When the strain was cultivated in the optimum culture medium containing ɛ-caprolactam as an inducer, in particular, the specific activity of NHase in the culture was 13 000 times higher than that without addition of amides, nitriles or acids. In this case, NHase formed accounted for 12% of the total cellular soluble protein. The purified NHase did not act on ɛ-caprolactam, and ɛ-caprolactam was not degraded during the cultivation by the strain, suggesting that ɛ-caprolactam seems to keep driving the NHase induction mechanism. Received: 3 March 1995/Received revision: 13 July 1995/Accepted: 7 September 1995     

Pedobacter jeongneungensis sp. nov., isolated from forest soil

Strain BH45(T) was isolated from forest soil of Mt. Bukhan in Jeongneung, Seoul, Korea. The Gram-staining-negative strain BH45(T) grows at 4-30°C (optimum of 25-30°C) and between pH 5-8 (optimum of pH 6-8). Its major cellular fatty acids are C(18:3) ω6c (6,9,12) and C(10:0). The G+C content of genomic DNA was 40.2 mol%. The major respiratory quinone system in strain BH45(T) is menaquinone-7. Phylogenetic analysis based on 16S rRNA gene sequences indicates that strain BH45(T) is closely related to the genus Pedobacter. Sequence similarities with P. terrae KCTC 12762(T), P. suwonensis KACC 11317(T), P. soli KACC 14939(T), P. alluvionis DSM 19624(T), P. roseus KCCM 42272(T), P. yonginense KCTC 22721(T) were 97.5, 97.1, 97.0, 97.0, 97.0, and 96.0%, respectively. DNA-DNA hybridization results distinguish strain BH45(T) from two Pedobacter species with high 16S rRNA gene sequence similarities. According to the phenotypic and molecular data, the strain BH45(T) clearly represents a novel species within the genus Pedobacter; thus, the name Pedobacter jeongneungensis sp. nov. is proposed for this strain. The type strain is BH45(T) (=KACC 15514(T) =JCM 17626(T)).     

Cometabolism of polychlorinated biphenyls: enhanced transformation of Aroclor 1254 by growing bacterial cells

Acinetobacter sp. strain P6 and a soil isolate, Arthrobacter sp. strain B1B, were tested for their ability to transform Aroclor 1254 as washed resting cells and as growing cells with biphenyl as the substrate. Growing cells were far superior to resting-cell suspensions in terms of total polychlorinated biphenyl (PCB) transformation, transformation of specific PCB congeners, and diversity of congeners that were attacked. Growing cells of Acinetobacter sp. strain P6 and Arthrobacter sp. strain B1B transformed 32 and 23% of the [14C]Aroclor 1254, respectively, whereas resting cells of the same respective cultures transformed only 17 and 8%. Transformation was significantly greater with resting cells in only 2 of 39 cases in which congeners were transformed by both growing and resting cells of both cultures. The components of 19 and 12 capillary gas-chromatographic peaks of Aroclor 1254 were transformed by biphenyl-grown resting cells of Acinetobacter sp. strain P6 and Arthrobacter sp. strain B1B, respectively, whereas the components of an additional 6 and 7 peaks were attacked by growing cells of the same respective cultures. Biphenyl oxidation by resting cells of both cultures decreased with time to less than 8% in 28 h. In addition to the normal 2,3-dioxygenase attack on PCBs, Acinetobacter sp. strain P6 also attacked congeners lacking an open 2,3-position. The ability of Acinetobacter sp. strain P6 to transform the components of 25 of the 40 largest peaks of Aroclor 1254 makes it one of the most versatile PCB-transforming organisms yet reported.     

Cometabolism of polychlorinated biphenyls: enhanced transformation of Aroclor 1254 by growing bacterial cells.

Acinetobacter sp. strain P6 and a soil isolate, Arthrobacter sp. strain B1B, were tested for their ability to transform Aroclor 1254 as washed resting cells and as growing cells with biphenyl as the substrate. Growing cells were far superior to resting-cell suspensions in terms of total polychlorinated biphenyl (PCB) transformation, transformation of specific PCB congeners, and diversity of congeners that were attacked. Growing cells of Acinetobacter sp. strain P6 and Arthrobacter sp. strain B1B transformed 32 and 23% of the [14C]Aroclor 1254, respectively, whereas resting cells of the same respective cultures transformed only 17 and 8%. Transformation was significantly greater with resting cells in only 2 of 39 cases in which congeners were transformed by both growing and resting cells of both cultures. The components of 19 and 12 capillary gas-chromatographic peaks of Aroclor 1254 were transformed by biphenyl-grown resting cells of Acinetobacter sp. strain P6 and Arthrobacter sp. strain B1B, respectively, whereas the components of an additional 6 and 7 peaks were attacked by growing cells of the same respective cultures. Biphenyl oxidation by resting cells of both cultures decreased with time to less than 8% in 28 h. In addition to the normal 2,3-dioxygenase attack on PCBs, Acinetobacter sp. strain P6 also attacked congeners lacking an open 2,3-position. The ability of Acinetobacter sp. strain P6 to transform the components of 25 of the 40 largest peaks of Aroclor 1254 makes it one of the most versatile PCB-transforming organisms yet reported.     

Molecular characterization and chromosomal localization of cytochrome P450 genes involved in the biosynthesis of cyclic hydroxamic acids in hexaploid wheat

The cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), are defensive secondary metabolites found in gramineous plants including wheat, maize and rye. cDNAs for five cytochromes P450 (P450s) involved in DIBOA biosynthesis (CYP71C6, CYP71C7v2, CYP71C8v2, CYP71C9v1 and CYP71C9v2) were isolated from seedlings of hexaploid wheat [( Triticum aestivum L. cv. Chinese Spring (2n=6x=42, genomes AABBDD)] by RT-PCR and screening of a cDNA library. CYP71C9v1 and CYP71C9v2 are 97% identical to each other in amino acid and nucleotide sequences. The cloned P450 species showed 76-79% identity at the amino acid level to the corresponding maize P450 species CYP71C1-C4, which are also required for DIBOA biosynthesis. The wheat P450 cDNAs were heterologously expressed in the yeast ( Saccharomyces cerevisiae) strain AH22. Microsome fractions from yeast cells expressing these P450 species catalyzed the same reactions as their maize orthologs. The chromosomes carrying the cyp71C6- C9v1 orthologs were identified by Southern hybridization using aneuploid lines of Chinese Spring wheat. The cyp71C9v1 orthologs were located on the chromosomes of wheat homoeologous group-4. The orthologs of the other P450 genes, cyp71C7v2, cyp71C6 and cyp71C8v2, were located on group-5 chromosomes. The same P450 genes were also present in the three ancestral diploid species of hexaploid wheat, T. monococcum (AA), Aegilops speltoides [BB (approximately SS)] and Ae. squarrosa (DD).     

Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X7 receptor-deficient mice

The P2X(7) receptor (P2X(7)R) is a two transmembrane receptor that is highly expressed on the surface of immune cells. Loss of function polymorphisms in this receptor have been linked to increased susceptibility to intracellular pathogens. P2X(7)R gene knockout (P2X(7)R(-/-); on a C57Bl/6J background), C57Bl/6J and BALB/c mice were infected with the avirulent ME49 strain of the intracellular parasite, Toxoplasma gondii, and susceptibility determined by monitoring weight loss. P2X(7)R(-/-) mice lost significantly more weight than C57Bl/6J mice from day 8p.i. C57Bl/6J, in turn, lost significantly more weight than BALB/c mice. Thus, by day 10p.i., P2X(7)R(-/-) mice had lost 5.7 ± 0.7% of their weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12p.i., P2X(7)R(-/-) mice had lost 15.1±0.6%, C57Bl/6J had lost 10.1±0.8% and BALB/c had lost 4.8 ± 0.8% of their weight. Neither parasite burden nor liver pathology was greater in the P2X(7)R(-/-) mice than in C57Bl/6J mice but BALB/c mice had significantly smaller numbers of parasites and less pathology in their livers than these strains. Absence of the P2X(7) receptor did not affect IFN-γ, IL-12, IL-1β, monocyte chemoattractant protein-1 (MCP-1) or TNF production. However, both P2X(7)R(-/-) and C57Bl/6J mice produced more IL-1β and TNF than BALB/c mice. There was one important point of differentiation between the P2X(7)R(-/-) and C57Bl/6J mice, namely the significantly enhanced and prolonged production of nitric oxide, accompanied by delayed production of IL-10 in the P2X(7)R-deficient mice.     

Effects of sporulation pH on the heat resistance and the sporulation of Bacillus cereus

Spores of Bacillus cereus ATCC 7004, 4342 and 9818 were obtained in nutrient agar at several pH from 5·9 to 8·3. The optimum pH for sporulation was around 7, but good production of spores was obtained in the range 6·5–8·3. With all three strains, D -values clearly dropped with sporulation pH, decreasing by about 65% per pH unit. z -Values were not significantly modified ( P > 0·05) by this factor. Mean z -values of 7·13 °C ± 0·16 for strain 7004, 7·67 °C ± 0·04 for 4342 and 8·80 °C ± 0·64 for 9818 were obtained.     

Pseudoalteromonas xishaensis sp. nov., isolated from Acanthaster planci in the Xisha islands

A Gram-negative, aerobic, motile by means of single polar flagellum, short rod-shaped marine bacterium, designated strain E418^T, was isolated from the spines on the body surface of starfish Acanthaster planci in the Xisha islands, China. Cells of strain E418^T were found to grow optimally at pH 7–8, at 25–37 °C, and in the presence of 2–5 % (w/v) NaCl. Phylogenetic analysis based on the comparison of 16S rRNA gene sequences revealed that strain E418^T is a member of the genus Pseudoalteromonas. The closest relative to this strain was found to be P. ruthenica LMG 19699^T, with a similarity level of 97.7 %. DNA relatedness between the novel isolate and this phylogenetically related species was 57.4 %. Strain E418^T decomposed Tween 80, gelatin, and casein, but was unable to decompose starch and grow on DNase Agar. The cellular fatty acid profile consisted of significant amounts of C16:1ω7c/C16:1ω6c, C18:1ω7c/C18:1ω6c, C16:0, and C17:1ω8c. The G+C content of DNA of this strain was determined to be 46.7 mol%. Phenotypic characteristics, phylogenetic analysis and DNA–DNA relatedness data suggest that strain E418^T represents a novel species of the genus Pseudoalteromonas, for which the name Pseudoalteromonas xishaensis sp. nov. is proposed. The type strain of P. xishaensis is strain E418^T (DSM 25588^T = NBRC 108846^T = CCTCC AB 2011177^T).     

Conversion of squid pen by Serratia ureilytica for the production of enzymes and antioxidants

Two proteases (P1 and P2) and a chitinase (C1) were purified from the culture supernatant of Serratia ureilytica TKU013 with squid pen as the sole carbon/nitrogen source. The molecular masses of P1, P2 and C1 determined by SDS-PAGE were approximately 50 kDa, 50 kDa and 60 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of P1, P2 and C1 were (pH 10, 40 degrees C, pH 7-11, and <50 degrees C), (pH 10, 40 degrees C, pH 8-11, and <40 degrees C) and (pH 6, 50 degrees C, pH 5-8, and <50 degrees C), respectively. P1 and P2 were inhibited by Mg(2+), EDTA and C1 was inhibited completely by Cu(2+). The antioxidant activity of TKU013 culture supernatant was 72% per mL (DPPH scavenging ability). With this method, we have shown that squid pen wastes can be utilized and have revealed its hidden potential in the production of functional foods.     

A two-protein component 7 alpha-cephem-methoxylase encoded by two genes of the cephamycin C cluster converts cephalosporin C to 7-methoxycephalosporin C.

Two genes, cmcI and cmcJ, corresponding to open reading frames 7 and 8 (ORF7 and ORF8) of the cephamycin C cluster of Nocardia lactamdurans encode enzymes that convert cephalosporin C to 7-methoxycephalosporin C. Proteins P7 and P8 (the products of ORF7 and ORF8 expressed in Streptomyces lividans) introduce the methoxyl group at C-7 of the cephem nucleus. Efficient hydroxylation at C-7 and transfer of the methyl group from S-adenosylmethionine require both proteins P7 and P8, although P7 alone shows weak C-7 hydroxylase activity and strong cephalosporin-dependent NADH oxidase activity. Both P7 and P8 appear to be synthesized in a coordinated form by translational coupling of cmcI and cmcJ. Protein P7 contains domains that correspond to conserved sequences in cholesterol 7 alpha-monooxygenases and to the active center of O-methyltransferases by comparison with the crystal structure of catechol-O-methyltransferase. Protein P8 may act as a coupling protein for efficient hydroxylation at C-7 in a form similar to that of the two-component system of Pseudomonas putida p-hydroxyphenylacetate-3-hydroxylase.     

无细胞百日咳疫苗效价检定用攻击菌液氮保存的稳定性

为了考察无细胞百日咳疫苗效价检定用攻击菌在液氮中保存的稳定性,对液氮保存1~7年的4~9代百日咳攻击菌随机抽取7~11批次样品,采用LD50检测并经统计学处理,分析其样品的毒力变化。试验结果显示,4~9代的百日咳攻击菌液氮保存5年,毒力仍能达到《中华人民共和国药典》三部(2005版)的要求;保存6年和7年,4~7代的百日咳攻击菌毒力仍符合上述要求,8、9代的攻击菌毒力的合格率分别为84%和40%。试验证实,无细胞百日咳效价检定用攻击菌液氮保存5年具有良好的稳定性,可用于无细胞百日咳疫苗的效价检定。