Molecular characterisation of the Stc mutation of Escherichia coli K-12

The previously described Stc ^- (suppressor of TolC) mutation modifies the phenotype of tolC mutants from OmpF⁻ to OmpF⁺. Restriction mapping of chromosomal DNA from Stc ⁺ and Stc⁻ strains was performed to investigate the nature of the mutation which was shown to be a deletion, upstream of the ompC gene. DNA from the region of the deletion was cloned into pUC18 and a 650-bp PstI-EcoRI fragment was sequenced. The deletion started 49 bp upstream of the AUG start codon of the ompC gene, thus removing part of the ompC promoter and the whole of the micF gene. We suggest that the deletion of micF gives rise to the Stc⁻ phenotype since the effect of micF expression is assumed to reduce ompF expression, and the Stc⁻ phenotype involves increase in ompF expression.     

Detection and quantification of phnE gene from oil-contaminated soil samples by competitive quantitative PCR

The phnE gene encoding catechol 2,3-dioxygenase belonging to the meta-cleavage pathway was selected as the marker gene and was detected and quantified from soil samples by competitive quantitative PCR. A PCR primer pair was designed based on the phnE gene to amplify the target DNA bands and competitor DNA bands. The phnE gene was detected in two samples of three. In samples S1 and S2, the phnE gene copy number was 6.2×10⁷/g soil and 5.8×10⁷/g soil, respectively. But no phnE gene was detected in sample S3. The target DNA bands were extracted and expressed. The results confirmed that the target DNA bands were the native phnE genes.     

DNA-biding of IQ, Me-IQ and Me-IQx, strong mutagens found in broiled foods

Non-covalent DNA-binding has been studied of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (Me-IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (Me-IQx), strong mutagens found in broiled foods. These mutagens are intercalated into DNA, as found by ultraviolet absorption gel electrophoresis. The binding of IQ is stronger with GC pairs than AT pairs in DNA. The binding constants with calf thymus DNA are 1.6 × 10⁶ (Me-IQ), 0.9 × 10⁶ (IQ) and 0.7 × 10⁶ M⁻¹ (Me-IQx) at pH 6.0. This order of DNA affinity agrees with the order of mutagenicity towards Salmonella typhimurium TA98.     

Effect of p53 genotype on gene expression profiles in murine liver

The tumor suppressor protein p53 is a key regulatory element in the cell and is regarded as the “guardian of the genome”. Much of the present knowledge of p53 function has come from studies of transgenic mice in which the p53 gene has undergone a targeted deletion. In order to provide additional insight into the impact on the cellular regulatory networks associated with the loss of this gene, microarray technology was utilized to assess gene expression in tissues from both the p53^−/− and p53^+/− mice. Six male mice from each genotype (p53^+/+, p53^+/−, and p53^−/−) were humanely killed and the tissues processed for microarray analysis. The initial studies have been performed in the liver for which the Dunnett test revealed 1406 genes to be differentially expressed between p53^+/+ and p53^+/− or between p53^+/+ and p53^−/− at the level of p ≤ 0.05. Both genes with increased expression and decreased expression were identified in p53^+/− and in p53^−/− mice. Most notable in the gene list derived from the p53^+/− mice was the significant reduction in p53 mRNA. In the p53^−/− mice, not only was there reduced expression of the p53 genes on the array, but genes associated with DNA repair, apoptosis, and cell proliferation were differentially expressed, as expected. However, altered expression was noted for many genes in the Cdc42-GTPase pathways that influence cell proliferation. This may indicate that alternate pathways are brought into play in the unperturbed liver when loss or reduction in p53 levels occurs.     

Effects of environmental factors, age and genotype on sperm production and semen quality in Bos indicus and Bos taurus AI bulls in Brazil

The effects of ambient temperature and humidity, month, age and genotype on sperm production and semen quality in AI bulls in Brazil were evaluated. Data from two consecutive years were analyzed separately. Seven Bos indicus and 11 Bos taurus bulls from one artificial insemination (AI) center were evaluated in Year 1 and 24 B. indicus and 16 B. taurus bulls from three AI centers were evaluated in Year 2. Ambient temperature and humidity did not significantly affect sperm production and semen quality, probably because there was little variation in these variables. Month accounted for less than 2% of the variation in sperm production and semen quality. Increased bull age was associated with decreased sperm motility (P<0.10) and increased minor sperm defects (P<0.001) in Year 1. B. indicus bulls had greater (P<0.005) sperm concentration than B. taurus bulls in both years (1.7×10⁹/ml versus 1.2×10⁹/ml in Year 1 and 1.6×10⁹/ml versus 1.2×10⁹/ml in Year 2, respectively). Ejaculate volume was not significantly affected by genotype in Year 1 (6.6 ml versus 6.9 ml in B. indicus and B. taurus bulls, respectively), but B. indicus bulls had greater (P<0.05) total (11.4×10⁹ versus 8.2×10⁹) and viable (6.7×10⁹ versus 4.9×10⁹) numbers of spermatozoa in the ejaculate than B. taurus bulls. In Year 2, B. taurus bulls had greater (P<0.05) ejaculate volume than B. indicus bulls (8.2 ml versus 6.7 ml, respectively) and total and viable number of spermatozoa in the ejaculate were not significantly different between genotypes (10.3×10⁹ versus 9.1×10⁹ and 6.1×10⁹ versus 5.4×10⁹ in B. indicus and B. taurus bulls, respectively). Sperm motility was not significantly affected by genotype (mean, 59%). In Year 1, B. indicus bulls tended (P<0.10) to have more major sperm defects and had more (P<0.05) total sperm defects than B. taurus bulls (11.8% versus 8.7% and 13.6% versus 10.0%, respectively). In Year 2, B. indicus bulls tended (P<0.10) to have more total sperm defects than B. taurus bulls (16.2% versus 13.3%, respectively). In conclusion, neither ambient temperature and humidity nor month (season) significantly affected sperm production and semen quality. B. indicus bulls had significantly greater sperm concentration and B. taurus bulls had significantly fewer morphologically defective spermatozoa.     

唾液酸免疫球蛋白型凝集素-15促进结直肠癌细胞增殖和迁移并抑制肿瘤组织中CD4+与CD8+T细胞浸润

唾液酸免疫球蛋白型凝集素-15(sialic acid-binding immunoglobulin-type lectin-15,Siglec-15)属于Siglecs家族的一员,是一种新型免疫抑制分子。Siglec-15在多种人类肿瘤细胞和肿瘤相关巨噬细胞中高表达,但Siglec-15在结直肠癌(colorectal cancer,CRC)中的生物学功能及其对免疫微环境的影响尚不明确。本文旨在分析Siglec-15异常表达对CRC细胞功能及CD4⁺T细胞、CD8⁺T细胞浸润的影响。首先,分析TCGA数据库中结直肠癌与正常组织中Siglec-15 mRNA表达水平,并对52例人CRC与配对癌旁组织进行免疫组织化学染色(IHC),发现Siglec-15在CRC中的表达水平高于癌旁组织(P<0.01)。CCK8和划痕愈合结果显示,敲低Siglec-15能抑制人CRC细胞SW480增殖(P<0.01)和迁移(P<0.05)。磁珠分选小鼠脾的CD8⁺T细胞并与小鼠CRC细胞MC38共培养,发现MC38细胞过表达Siglec-15能抑制CD8⁺T细胞对其的杀伤以及IFN-γ和TNF-α的分泌(P<0.01)。小鼠荷瘤结果表明,过表达Siglec-15可以促进小鼠肿瘤生长(P<0.05)。单样本基因集富集分析、荷瘤小鼠肿瘤组织及人结直肠癌组织IHC分析均表明,Siglec-15高表达时,肿瘤微环境中CD4⁺T细胞、CD8⁺T细胞浸润减少(P<0.05)。综上所述,Siglec-15可能通过促进CRC细胞增殖迁移以及抑制CD4⁺T细胞、CD8⁺T细胞浸润促进结直肠癌进展。本文为探究Siglec-15在CRC中的免疫抑制作用提供了一些新的实验依据。     

Detection of Mycobacterium immunogenum by real-time quantitative Taqman PCR

A quantitative real-time 5′-nuclease (Taqman) PCR technique was developed to specifically detect Mycobacterium immunogenum. rpoB-specific primers and Taqman probe were evaluated for detection of M. immunogenum DNA extracted from pure cultures and from industrial metal working fluids (MWFs). Specificity was confirmed and the sensitivity of detection of M. immunogenum genomic DNA was shown to be approximately 9 fg (2 cell equivalents). When tested on industrial metal working fluids from the UK and USA from which no M. immunogenum CFU were recovered, the assay detected between 3.4 × 10¹ and 1.9 × 10⁴ cell equivalents (CE) per ml, and increased the detection rate over culture to 37.5% (12 of 32 samples). This assay provides a specific, sensitive and rapid method for the detection of M. immunogenum and is applicable within industry for the early detection of this human pathogen and to the possible prevention of hypersensitivity pneumonitis (HP) in workers.     

Relationship between cyprid energy reserves and metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica)

Nauplii batch cultures of Balanus amphitrite were reared with four different diatoms (Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, silicate-limited C. gracilis) at three different cells concentrations: 1×10⁵, 5×10⁵, and 1×10⁶ cells ml⁻¹. The cyprid energy reserves were quantified as the ratio of triacylglycerols (TAG) to DNA. Energy reserves of larvae fed on different diatoms at a concentration of 1×10⁶ cells ml⁻¹ were ranked in the order: silicate-limited C. gracilis>C. gracilis>T. pseudonana>S. costatum. There was a significant linear relationship between the TAG content of the diet and cyprid energy reserves. The effect of cyprid energy reserves on metamorphosis to polystyrene surface in the presence and the absence of conspecific settlement factor (SF) was studied after 12, 24, and 48 h of incubation. A strong positive correlation between energy reserves and percent metamorphosis was observed in the absence of SF (r_{12 h}=0.88, r_{24 h}=0.82, r_{48 h}=0.68, P<0.05). A weak positive correlation was observed in the presence of SF (r_{12 h}=0.43, r_{24 h}=0.48, r_{48 h}=0.50, P<0.05). In both treatments, more than 80% of the cyprids with high energy reserves metamorphosed within 24 h. In contrast, a high proportion of cyprids with low energy reserves metamorphosed in response to SF in 24 h. Our results indicate that discriminatory metamorphic behavior of cyprids is closely linked to their TAG/DNA ratio, a proxy for energy reserve.     

Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults

We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2+3 deletions and t(14;18) translocations as a measure of illegitimate V(D)J recombination. We determined the baseline frequencies of these two mutations in mononuclear leukocyte DNA from the umbilical cord blood of newborns and from the peripheral blood of adults. In an initial group of 21 newborns, no t(14;18) translocations were detected (<0.049×10⁻⁷). The frequency of HPRT exons 2+3 deletions was 0.10×10⁻⁷ per mononuclear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%–70%) and previous results using the T-cell cloning assay (2–3×10⁻⁷ per clonable T-cell). Phytohemagglutinin (PHA), as used in the T-cell cloning assay, was examined for its effect on the frequencies of these mutation events in mononuclear leukocytes from an additional 11 newborns and from 12 adults. There was no significant effect of PHA on t(14;18) translocations which were rare among the newborns (1 detected among 2.7×10⁸ leukocytes analyzed), and which occurred at frequencies from <1×10⁻⁷ (undetected) to 1.6×10⁻⁴ among the adults. The extremely high frequencies of t(14;18)-bearing cells in three adults were due mainly to in vivo expansion of two to six clones. However, PHA appeared to stimulate a modest (although not significant) increase in the frequency of HPRT exons 2+3 deletions in the leukocytes of the newborns, from 0.07×10⁻⁷ to 0.23×10⁻⁷. We show that both the direct PCR assay and the T-cell cloning assay detect similar frequencies of HPRT exons 2+3 deletions when calculations are normalized to blood volume, indicating that the apparent discrepancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effects of environmental genotoxic agents on this clinically important recombination mechanism.     

Acridinium ester conjugated to lectin as chemiluminescent histochemistry marker

Cell differentiation/dedifferentiation includes changes in oligosaccharide composition and distribution in the cell surface glycoconjugates. Lectins have been used as auxiliary tools in histopathological diagnosis of mammary, uterus and brain pathologies. Acridinium ester (AE) conjugated to biomolecules has been employed in chemiluminescent analytical applications. This work aimed to use a lectin, concanavalin A (Con A), conjugated to AE as a chemiluminescent histochemistry tool. Biopsies of normal and infiltrating duct carcinoma (IDC) of mammary tissues were treated by a Con A-AE derivative. Photon emission, observed during the breakage of the chemical bound between Con A and AE, was quantified, expressed in relative light units (RLU) and correlated to the labelling of the normal and transformed tissues. The results demonstrated that RLU presented a linear relationship with the labelled tissue area in the range 0.125-1.0 cm² (r=0.98). Furthermore, RLU was much higher for the IDC (1283.920×10³±220.621×10³) than the normal tissue (2.565×10³±0.247×10³), namely, about 500 times higher. The Con A-AE conjugation efficiency, differential staining of normal and IDC tissues, and quantification of results contribute to a decrease in the subjectivity in routine histopathological diagnoses and indicate that acrydinum ester can join other lectin marker to be used in histochemistry.     

Microbial characterization during composting of municipal solid waste

This study investigates the prevailing physico-chemical conditions and microbial community; mesophilic bacteria, yeasts and filamentous fungi, bacterial spores, Salmonella and Shigella as well as faecal indicator bacteria: total coliforms, faecal coliforms and faecal Streptococci, present in a compost of municipal solid waste. Investigations were conducted in a semi-industrial pilot plant using a moderate aeration during the composting process. Our results showed that: (i) auto-sterilization induced by relatively high temperatures (60–55°C) caused a significant change in bacterial communities. For instance, Escherichia coli and faecal Streptococci populations decreased, respectively, from 2×10⁷ to 3.1×10³ and 10⁷ to 1.5×10³ cells/g waste dry weight (WDW); yeasts and filamentous fungi decreased from 4.5×10⁶ to 2.6×10³ cells/g WDW and mesophilic bacteria were reduced from 5.8×10⁹ to 1.8×10⁷ bacteria/g WDW. On the other hand, the number of bacterial spores increased at the beginning of the composting process, but after the third week their number decreased notably; (ii) Salmonella disappeared completely from compost by the 25th day as soon as the temperature reached 60°C; and (iii) the bacterial population increased gradually during the cooling phase. While Staphylococci seemed to be the dominant bacteria during the mesophilic phase and at the beginning of the thermophilic phase, bacilli predominated during the remainder of the composting cycle. The appearance of gram-negative rods (opportunistic pathogens) during the cooling phase may represent a serious risk for the sanitary quality of the finished product intended for agronomic reuse. Compost sonication for about 3 min induced the inactivation of delicate bacteria, in particular gram-negatives. By contrast, gram-positive bacteria, especially micrococcus, spores of bacilli, and fungal propagules survived, and reached high concentrations in the compost.     

Biological Control and Use of Adjuvants against Multiple Seeded Cocklebur (Xanthium strumarium) in Comparison with Several Other Cocklebur Types

Common cocklebur has several biotypes including multiple seeded cocklebur (MSC), NCC-TX, and NCC-MS. Alternaria helianthi applied at 2.5×10⁴ conidia mL^-1 in a 50% micro-emulsion of unrefined corn oil (MESUCO) or 0.2% Silwet L 77 caused 60-75% mortality on NCC-TX and MSC. Increasing the conidial concentration to 5×10⁴ mL^-1 increased mortality to 100% on MSC and NCC-TX, and 75% on NCC-MS. At 10×10⁴ conidia mL^-1, A. helianthi caused 100% mortality in all three biotypes. No mortality occurred in any biotype at inoculation rates of 2.5 and 5×10⁴ conidia mL^-1 when applied in water. Increasing the dew period from 0 to 12 h increased mortality from 0 to 100% on all three biotypes at a rate of 2.5×10⁴ conidia mL^-1 in Silwet and MESUCO. MSC appears to be the most sensitive biotype.     

肝脏特异性CD36基因敲除小鼠的制备及鉴定

目的:运用Cre/Loxp重组酶系统构建肝脏特异性CD36基因敲除小鼠并进行鉴定和验证,为研究CD36的生物学功能奠定基础。方法:构建CD36打靶载体,电转转染胚胎干细胞,通过长链PCR筛选出正确同源重组的阳性克隆,阳性胚胎干细胞克隆经扩增后,注射入C57BL/6J小鼠的囊胚中,获得嵌合小鼠,再与Flp小鼠交配筛选获得Flox杂合子小鼠,该小鼠与引进的Alb-Cre小鼠交配,在F3代获得CD36fl/fl:Alb-Cre+基因型小鼠,即为肝脏特异性CD36敲除小鼠。采用PCR鉴定小鼠基因型,PCR、实时荧光定量PCR和Western blot验证小鼠肝脏CD36敲除效果,Western blot检测小鼠肾脏、脂肪和心肌组织CD36表达情况,HE染色观察小鼠肝脏形态学改变。结果:建立了CD36基因的Flox杂合子小鼠,与Alb-Cre小鼠交配后,在F3代筛选出CD36fl/fl:AlbCre-和CD36fl/fl:Alb-Cre+基因型小鼠,DNA水平证实CD36fl/fl:Alb-Cre+基因型小鼠肝脏CD36基因通过Cre/Loxp重组酶系统被敲除。与CD36fl/fl:Alb-Cre-基因型小鼠相比,CD36fl/fl:Alb-Cre+基因型小鼠肝脏CD36mRNA和蛋白表达水平显著降低,肾脏、脂肪和心肌组织CD36蛋白表达无差别,肝脏形态学特征无明显差异。结论:通过Cre/Loxp重组酶系统成功构建了肝脏特异性CD36基因敲除小鼠,为研究CD36在肝脏代谢和肝脏疾病中的功能提供了动物模型。     

Catalysis of plastocyanin electron self-exchange by redox-inert multivalent cations

Electron self-exchange in solutions of the ‘blue’ copper protein plastocyanin is catalysed by the redox-inert multivalent cations Mg²⁺ or Co(NH₃)³⁺₆. Measurements of specific ¹H-NMR line broadening with 50% reduced solutions in the presence of these cations show that electron exchange proceeds through encounters of cation-protein complexes which dissociate at high ionic strength. In the presence of 8mM (5 equivalents/total protein) Co(NH₃)³⁺₆, with 10 mM cacodylate (pH^*6.0) as background electrolyte, the bimolecular rate constant at 25°C is 7 × 10⁴ M⁻¹·s⁻¹. For comparison, the ‘electrostatically screened’ rate constant measured in 0.1 M KCl in the absence of added multivalent cations is ˜ 4 × 10³ M¹·s⁻¹.

Plastocyanin Electron self-exchange NMR Protein-protein interaction Multivalent cation Blue copper protein     

Structure, physical, chemical and photophysical properties of Pt(bph) (CO)2, where bph is the biphenyl dianion

The complex Pt(bph) (CO)₂ crystallizes in the space group Cmcm with a = 18.647(6), B = 9.566(2) and C = 6.4060(5) Å. The geometry of the molecule is slightly distorted from square planar with a Pt---C(CO) bond distance of 1.98(2) Å and a Pt---C(bph) bond distance of 2.04(2) Å. The Pt(bph)(CO)₂ complex serves as a precursor for the preparation of a wide variety of Pt(bph)X₂ complexes, where X = monodentate ligands such as acetonitrile, pyridine, etc., and X₂ = bidentate ligands such as bypyridine, 1,10-phenanthroline, etc. In the solid state, the complex exhibits a green color, but when ground with an alkali metal salt turns deep blue to purple. In CH₂Cl₂, the color disappears but optical transitions are observed at 271 nm (2.7 × 10⁴ M⁻¹ cm⁻¹), 303 nm (1.1 × 10⁴ M⁻¹ cm⁻¹) and 330 nm (5.5 × 10³ M⁻¹ cm⁻¹). The complex is a weak emitter exhibiting a structured spectrum in CH₂Cl₂ at r.t. with maxima located at 562 and 594 nm and an emission lifetime of 3.1 μs when excited at 337 nm.     

Kinetics of laccase-catalysed TEMPO oxidation

The oxidation of TEMPO (2,2,6,6-tetramethyl-piperidine-1-oxyl radical) has been studied in the presence of recombinant laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) from Polyporus pinsitus (rPpL), Myceliophthora thermophila (rMtL), Coprinus cinereus (rCcL) and Rhizoctonia solani (rRsL) in buffer solution pH 4.5–7.3 and at 25 °C. At pH 5.5 the oxidation constant calculated from the initial rate of TEMPO oxidation was 1.7 × 10⁴, 1.4 × 10³, 7.8 × 10² and 5.2 × 10² M⁻¹ s⁻¹ for rPpL, rRsL, rCcL and rMtL, respectively. The maximal activity of rPpL-catalysed TEMPO oxidation was at pH 5.0. The pK_a obtained in neutral pH range was 6.2. The reactivity of laccases is in a good agreement with laccases copper type I redox potential.

TEMPO oxidation rate increased 541 times in the presence of 10-(3-propylsulfonate) phenoxazine (PSPX). The model of synergistic TEMPO and PSPX oxidation was proposed. Experimentally obtained rate constants for rPpL-catalysed PSPX oxidation were in a good agreement with those calculated from the synergistic model, therefore confirming the feasibility of the model. The acceleration of TEMPO oxidation with high reactive laccase substrates opens new possibilities for TEMPO application as a mediator.     

Influence of Agricultural By-products in Liquid Culture on Chlamydospore Production by the Potential Mycoherbicide Fusarium oxysporum Foxy 2

Economically feasible inoculum mass production methods are required for successful application of Fusarium oxysporum Foxy 2 as a potential mycoherbicide. Therefore, different substrates (agricultural by-products) and the factors that influence the production of spores, especially chlamydospores, of Foxy 2 were investigated in liquid cultures. The substrates tested were cotton seed cake, maize stover, wheat and triticale stillage. The presence of plant fibers in the medium of unfiltered cotton seed cake (2.5%, w/v) significantly enhanced chlamydospore, micro- and macroconidia production by 150, 185, and 300%, respectively, compared to the filtered (fiber-free) medium. Regardless of the type of substrate tested, Foxy 2 was able to produce abundant chlamydospores (0.14-2.7×10⁷ mL^-1) in all growth media. Generally, increasing the concentrations of cotton seed cake and maize stover in the medium significantly increased chlamydospore formation; however, this was not the case for wheat-based stillage. To optimize conditions required for chlamydospore production of Foxy 2, the effect of near ultra-violet (NUV) light, substrate combinations (synergism), level of agitation, incubation time and their interaction were studied. A liquid culture of 2.5% (w/v) filtered cotton seed cake, exposed to continuous NUV for 15 days, doubled the yield of chlamydospores (4.7×10⁶ mL^-1) and macroconidia (5×10⁵ mL^-1), and increased microconidial production by one-third (1×10⁸ mL^-1) compared to natural light. An apparent synergistic effect of substrate combination was observed, since the addition of 20% (v/v) of either wheat or triticale-based stillage to maize stover medium (1%, w/v) increased the number of chlamydospores produced by 16 or 12 times, respectively, compared to maize stover alone (1.4×10⁶ mL^-1), and 2 times more than either of the stillages. A significantly positive effect between a high level of agitation tested during incubation and chlamydospore production of Foxy 2 was recorded. All in all, substantial chlamydospore production of Foxy 2 (4.3×10⁷ mL^-1) was successfully achieved within 12 days of incubation in a single-step liquid fermentation through the combination of 0.5% (w/v) maize stover plus 20% (v/v) wheat-based stillage and a high level of agitation (200 rpm).     

AAV载体介导的蓬佩病模型小鼠体内基因治疗研究*

Objective: Pompe disease is a lysosomal glycogen storage disease caused by acid α-glucosidase (GAA) deficiency, which is characterized by glycogen accumulation in the heart, skeletal muscle, and central nervous system (CNS). AAV vector-mediated gene therapy is expected to be a breakthrough in the treatment of Pompe disease. In this study, AAV9 vector was used to mediate GAA gene transfer in Pompe disease model mice, and the changes of GAA protease activity, glycogen accumulation in tissues and pathological changes in mice after transgenic intervention were evaluated. Methods: Codon optimized GAA gene (coGAA) was carried by AAV9 vector, and the AAV vector was packaged by baculovirus production process. Adult Pompe model mice were given a single intravenous injection at the dose of 1.1×10¹³, 3.0×10¹³, 1.2×10¹⁴ vg/kg, and aged Pompe model mice were given a single intravenous injection at the dose of 3.0×10¹³ vg/kg. After reaching the end point of the experiment, the mice were euthanized, GAA protease activity was determined by fluorescence spectrophotometry, glycogen accumulation was observed by PAS staining, and pathological changes were detected by HE staining. Results: Five weeks after administration, GAA protein was widely expressed in all tissues of adult model mice, with higher expression levels in heart and liver, and lower expression levels in brain and spinal cord. After rAAV9-coGAA treatment, glycogen content in myocardium, skeletal muscle and brain decreased, and vacuolar degeneration in myocardium and skeletal muscle decreased significantly. After treatment, the tissue enzyme activity of the aged animals was significantly increased compared with that of the model mice. The vacuolar degeneration and inflammatory cell infiltration of the myocardium were decreased, but the pathological improvement of skeletal muscle was limited. Conclusion: A single intravenous injection of rAAV9-coGAA can enhance GAA enzyme activity, reduce glycogen accumulation and improve pathology in Pompe model mice. The therapeutic effect was dose-dependent, and the injection also had certain therapeutic effect on aged animals. This study laid a theoretical foundation for the clinical application of AAV9 mediated gene therapy via intravenous route in Pompe disease.