Cloning, sequencing and expression of a swine IgG H chain gene inE. coli

A DNA fragment about 1.5 kb has been isolated from spleen of adult Chinese swine by RT-PCR. The DNA fragment encodes immunoglobulin IgG H chain gene. Sequencing analysis showed that the DNA fragment is 1 425 bp long, complete CDS. The C region of the gene has been classified as Subclass Ig γ3, and is the same as reported by Sun et al., but V region of the present gene is 42 bp less by comparison. The gene has been ligated into expression vector pET-3b (NSEB)( - ). A protein about 52 ku has been expressed in E. coli with an expression level of about 21 % .     

Determination of specific surfaces of silica xerogets by SAXS

Small angle X-ray scattering (SAXS) with synchrotron radiation as X-ray source has been used to study the structure of silica xerogets prepared by sol-get process. Both the agreement of SAXS profiles with and the deviation from Porod’s law and Debye’s theory have been found, showing that there are differences between the structures of these xerogets. The specific surfaces of the samples whose SAXS profiles agreed with Porod’s law and Debye’s theory have been determined by analyzing SAXS data according to the methods of Porod and Debye, respectivety, and the results of both methods used were found to be similar. We have proposed the corresponding Porod and Debye analysis methods to determine the specific surfaces of samples whose SAXS profiles do not agree with Porod’s law and Debye’s theory, i.e. the negative or positive deviation. The results of both methods used here were also found to be close to each other. The specific surfaces fell between approximatety 80–150 m²/cm³ for the samples prepared under various conditions.     

Expression of human hepatitis C virus core antigen in tobacco plants by tobacco mosaic virus-based vector system

Tobacco mosaic virus (TMV) has the potential to highly express foreign gene. A novel TMV-basedin trans expression system was constructed. A TMV mutant TSHc had its coat protein replaced with hepatitis C virus (HCV) core antigen gene. Anotherr TMV mutant TSBD was replicase-defective. Coinfection of the two mutants could cause systemic infection in tobacco plants byin trans complementation of their functions. TSHc could effectively replicate and assemble to viral particles, which were a little longer than that of wild-type TMV. HCV core antigen was expressed in whole tobacco plants. A similar expression level of HCV core antigen was detected on serial passages, which suggested that this viral expression system be stable.     

Regularities of formation of binary intermetallic compounds between transition and non-transition elements

A four-parameter model based on the extended Miedema’s cellular model of alloy phases and pattern recognition methods has been used to study the regularities of the formation of binary intermetallic compounds between transition element and non-transition element. The formation criterion can be expressed as some inequities of electronegativity ∮, the valence electron density in Wagner-Seitz cell n_ws^1/3, Pauling’s metallic radius R and the number of valence electrons in atom Z or their functions. According to these empirical criterions, the “unknown” binary alloy system can be predicted, the predicted result is better than that of Miedema’s two-parameter model.     

鸡新城疫病毒HN蛋白多表位抗原的表达与免疫原性研究

有效抗原及表位的预测和筛选是疫苗研究的基础,在对鸡新城疫病毒HN蛋白抗原表位预测的基础上,对多表位抗原进行表达与免疫原性测定。根据生物信息学表位预测方法获得的家禽新城疫病毒抗原表位,利用PCR技术合成基因,构建pBVIL1-HN重组载体,转化大肠杆菌HB101,进行基因工程表达;经纯化蛋白后免疫小鼠,抗体滴度用酶联免疫吸附方法测定,确定抗原的免疫原活性。结果表明,多表位抗原基因经测序结果正确,融合基因在大肠杆菌得到高效表达,电泳纯融合多表位抗原经三次免疫得到抗血清,抗体滴度为1：8000。鸡新城疫病毒HN蛋白多表位抗原得到高效表达,且具有良好的免疫原性。     

t-bit semiclassical quantum Fourier transform

Because of the difficulty of building a high-dimensional quantum register,this paper presents an implementation of the high-dimensional quantum Fourier transform(QFT)based on a low-dimensional quantum register.First,we define the t-bit semi- classical quantum Fourier transform.In terms of probability amplitude,we prove that the transform can realize quantum Fourier transformation,illustrate that the requirement for the two-qubit gate reduces obviously,and further design a quantum circuit of the transform.Combining the classical fixed-window method and the implementation of Shor’s quantum factorization algorithm,we then redesign a circuit for Shor’s algorithm,whose required computation resource is approximately equal to that of Parker’s.The requirement for elementary quantum gates for Parker’s algorithm is 3 O (logN),and the quantum register for our circuit re- quires t-1 more dimensions than Parker’s.However,our circuit is t2 times as fast as Parker’s,where t is the width of the window.     

Identification of a disulfide bonded protein from cytoplasm ofBacillus subtilis

Conclusion In this study, P23 was found to be a disulfide-bonded cytoplasmic protein, abundant in late exponential phase and stationary phase cells, and was hardly detected in early exponential phase cells, the cells of sporulation process and spores. Therefore, synthesis of P23 was regulated by some specific factors or/and cellular environment. Conclusively, cytoplasm has a mechanism to catalyze the formation of disulfide bonds, which is consistent with Dermanet al. ’s conclusion from mutation experiment^[1].     

Prophylaxis of tumor through oral administration of IL-12 GM-CSF gene carried by live attenuated salmonella

A live attenuated AraA^- autotrophic mutant ofSalmonella typhimurium (SL3261) was used as carrier for eukaryotic expression vectors EGFPN1, pCMVmIL-12, pCMVhIL-12, pCMVmGM-CSF and pCMVhGM-CSF and was administered orally to BALB/c and C57BL/6 mice. After 6 weeks, these mice were challenged with 4T1 and Lewis tumor cells respectively. GFP expression and gene integration could be detected in mice’s livers, spleens, intestines, kidneys and tumors. The serum level of cytokines increased significantly in treated mice, so did the ratio of CD ₈ ⁺ /CD ₄ ⁺ , which resulted in the tumor regression and prolongation of the survival time of those mice. These researches laid an experimental foundation for the tumor gene therapy using live attenuated salmonella.     

Expression of E1 gene of a hepatitis C virus inE. coli and protein purification

The cDNA containing full encoding region of E1 antigen of HCV was cloned into an expression plasmid pRSETHisB. The recombinant plasmid pRSETE1 was introduced into the BL21 (DE3) strain ofE. coli. The engineering bacteria harbouring the pRSETE1 was cultivated in 2YT medium at 37°C. When the Expression of E1 protein was induced by 1 mmol IPTG, the bacteria was killed and the number of living cell was droped down from 10⁷ to 10³ cell/mL one hour post induction. Suggest that E1 protein is poisoned toE. coli. However, the 26kD polypeptide of E1 fussion protein still synthesized in appropriate condition. The expression level was about 10% of total protein 4 h after inducing. The E1 protin was purified by Ni²⁺-NTA-Agarose column chromatography to homogeneous. The purified E1 protein was sensitive and specific in reaction with anti-HCV antibody in sera. Supported by the Science Committec of Hubei Province Ye Linbai: born in Feb. 1948. Professor     

Construction of the glucose isomerase deficient strain ofStreptomyces M1033 by homologous recombination

After the establishment of the transformation conditions ofStreptomyces diastaticus No.7 Strain M1033, the integration plasmid pXW for homologous recombination, which contains a 600 bp fragment of incompleteGI (G138P. G247D) gene, has been constructed in order to realize the stable overexpression of theGI (G138P. G247D) which is valuable for large-scale industrial production. The Gigene’s disruption has been realized by pXW’s integration into M1033 chromosomes via homologous recombination andGI deficient strain ofStreptomyces M1033 has been obtained. The reliability of introduction of mutation has been proved by analysis of recombinant fragment and affirmance of existence of the mutation, as well as detection of the stability of the deficient strain.     

Rapid growth cost in ＂all-fish＂ growth hormone gene transsenic carp： Reduced critical swimming speed

Evidence has accumulated that there is a trade-off between benefits and costs associated with rapid growth. A trade-off between growth rates and critical swimming speed （Uc,t） had been also reported to be common in teleost fish. We hypothesize that growth acceleration in the F3 generation of ＂all-fish＂ growth hormone gene （GH） transgenic common carp （Cyprinus carpio L.） would reduce the swimming abilities. Growth and swimming performance between transgenic fish and non-transgenic controls were compared. The results showed that transgenic fish had a mean body weight 1.4--1.9-fold heavier, and a mean specific growth rate （SGR） value 6%-10% higher than the controls. Transgenic fish, however, had a mean absolute Ucr, （cm/s） value 22% or mean relative Ucrit （BL/s） value 24% lower than the controls. It suggested that fast-growing ＂all-fish＂ GH-transgenic carp were inferior swimmers. It is also supported that there was a trade-off between growth rates and swimming performance, i.e. faster-growing individuals had lower critical swimming speed.     

Cloning and expression analysis ofMBLL cDNA

Thembl (muscleblind) gene ofDrosophila encodes a nuclear protein which contains two Cys₃His motifs. The mutation ofmbl gene will disturb the differentiation of all theDrosophila’s photoreceptors. Primers have been designed according to human EST086139, which is highly homologous tombl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designatedMBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys₃His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology toDrosophila’s mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show thatMBLL is a widely expressed gene, but the expression amounts differ in these tissues.     

Speeding up implementation for Shor’s factorization quantum algorithm

In this paper, based on the implementation of semiclassical quantum Fourier transform, we first propose the concept of generation vector of ternary binary representation, construct the generation function’s truth table, prove that the generation vector of ternary binary representation is one kind of k ’s NAF representation and further find that its number of nonzero is not more than [(⌈log k⌉ + 1)/2]. Then we redesign a quantum circuit for Shor’s algorithm, whose computation resource is approximately equal to that of Parker (Their requirements of elementary quantum gate are both O(⌈logN⌉³), and our circuit requires 2 qubits more than Parker’s). However, our circuit is twice as fast as Parker’s.     

龙匙片降血糖作用的实验研究

【目的】龙匙方是广西壮族自治区人民医院俞祝全医生的临床经验方,本文研究其对正常小鼠及几种糖尿病模型小鼠降血糖的作用。【方法】采用链脲佐菌素致糖尿病、肾上腺素致高血糖、大剂量口服葡萄糖致高血糖小鼠模型,分别观察龙匙片对正常和糖尿病模型小鼠血糖的影响。【结果】高、中剂量组(11500mg·kg~(-1)·d~(-1)、5750mg·kg~(-1)·d~(-1))龙匙片对链脲佐菌素致糖尿病、肾上腺素致高血糖、大剂量口服葡萄糖致高血糖小鼠的血糖具有明显的降低作用;但高、低剂量(11500mg·kg~(~(-1))·d~(-1)、3060mg·kg~(-1)·d~(-1))龙匙片对正常小鼠血糖水平均无明显影响(P0.05)。【结论】龙匙片对糖尿病模型小鼠具有降低血糖的作用。     

A tumor suppressor genefhit prokaryotic expression and identification

The recombinant expression vector pGEMD-fhit which contains full encoding region offhit gene was constructed. The recombinant was introduced into the BL21 (DE3) strain ofE. coli and induced by 1 mmol/L IPTG to express a 29×10³ polypeptide offhit fusion protein. And the 29×10³ protein was sensitive and specific in reaction with anti-fhit antibody in Western blot. Foundation item: Supported by the National Natural Science Foundation of China (39770373) Biography: SUN Yan (1975−), female, Master of science, Research direction: gene engineering     

Efficient transfer and expression of human clotting factor IX cDNA in neonatal hemophilia B mice mediated by VSV-G pseudotyped retrovirus

The feasibility of in vivo gene therapy for hemophilia B by VSV-G pseudotyped retroviral vector was introduced. The novel packaging cell line 293GPG was used to produce VSV-G/G1NaBAIX pseudotyped virus with the highest titers up to 8.5×10⁸ cfu·mL^-1. In contrast to the conventional retrovirus, VSV-G pseudotyped virus was more resistant to inactivation by serum complements (P＜0.001). Our results also demonstrated that VSV-G pseudotyped virus was more stable in neonatal mice serum than in adult mice serum (P＜0.01). After intraperitoneal injection of different doses of virus, hFIX antigen was detected and lasted for more than 120 d, the highest level reached (72.5±6.1) ng·mL^-1. Moreover, the functional activity was improved to some extent in all hFIX-treated mice, the most remarkable improvement was observed in the mice treated with higher dose of virus whose clotting activity increased to (3.4±1.5)% and APTT (activated partial thromboplastin time) reduced to (43.2±7.2) s. The anti-hFIX antibody was not detected by the method of Bethesda, no germ line transmission and any side effects associated with gene transfer were found. Our results indicated that neonatal gene therapy for hemophilia B mice by VSV-G pseudotyped retrovirus is promising.     

Cloning, construction of prokaryotic expression vector and expression ofEscherichia coli cytosine deaminase gene

Cytosine deaminase gene ofEscherichia coli strain H-30 was cloned, and its initiation codon of ‘GTG’ was mutated to ‘ATG’ by PCR. Prokaryotic recombinant expression vector pBV220-CD was constructed. Clone with high enzyme activity were selected by detecting their specific activity of cytosine deaminase. 5-FC(5-FC, 5-fluorocytosine) could induce the lethal toxicity to cells containing active CD gene. DNA sequence analysis indicated that there were 16 altered bases and 5 of them resulted in the alteration of amino acids in predicted peptide by comparing DNA sequence of the clone H-30-CD-11 with high enzyme activity with CD gene reported in Gene Bank.     

Delayed resistance to Cucumber mosaic virus mediated by 3’UTR-derived hairpin RNA

RNA silencing has been shown to function in the plant antivirus defense response, leading to viral RNA degradation induced by vsiRNA-containing RISC cleavage activity. Cucumber mosaic virus （CMV） 3′UTR sequences share a high conservation of nucleotide sequence and secondary structures that are important for CMV replication. Here, in an attempt to simultaneously target the multiple genomic and subgenomic RNAs of CMV for degradation, CMV 3′UTR were used to design hairpin RNA （hpRNA） to transform tobacco （Xanthi. nc） so as to constitutively produce viral siRNAs. Most of the transgenic plants expressing CMV Q strain （Q-CMV, subgroup Ⅱ strain） RNA3 3′UTR-derived hpRNA showed delayed resistance to Q-CMV infection and exhibited recovery phenotypes. Compared with Q-CMV-inoculated leaves, the upper leaves showed weak or no disease symptoms and a reduced accumulation level of viral RNAs. Together with transient assays, our results indicate that the 3′UTR-derived siRNAs were biologically active in targeting viral RNA for degradation. Recovery resistance in transgenic plants was also observed against subgroup IB strain SD-CMV infection, indicating a broad-spectrum anti-CMV effect of the 3′UTR-based antiviral silencing. Northern blot assays indicated that there was no strong correlation between the degree of resistance and the accumulation level of 3′UTR-derived siRNAs, suggesting that to target a highly structured RNA, such as the CMV 3′UTR, the quantity of siRNAs may not be the only determinant of silencing efficiency. Target RNA secondary structures may also affect target accessibility, siRNA-containing RISC-target recognition and the consequent antiviral effect.     

JAK3 mediatesc-myc gene expression induced by interleukin-2

C myc gene expression can be rapidly induced by IL 2 through intracellular signal transduction which is triggered by the interaction between IL 2 and its receptor (IL 2R). JAK3 which associates to the intracellular domain of IL_2R γ may play a critical role in this process. To reveal the action of JAK3 in c myc induction, a chimeric receptor gene IL_2R α/γ/Δ NJAK3 is constructed which consists of extracellular domain derived from IL 2R α subunit (IL_2R α), the transmembrane sequence derived from IL_2R γ and the cytoplasmic domain derived from the catalytic domain of JAK3 (Δ NJAK3), and then transfected this chimeric gene into mouse fibroblast cell line L929 β which had been transfected with IL_2R β gene and stably expressed IL_2R β in high level. In the transfectants coexpressing IL_2R β and α/γ/Δ NJAK3, the stimulation of IL_2 could intensively induce c myc gene expression. Because the whole cytoplasmic domain of IL_2R γ which could recruit signaling molecules was replaced by JAK3 and the c myc could be still induced by IL_2 in this situation, the results here gave the direct evidence demonstrating that JAK3 plays an important role in c myc gene expression induced by IL_2.