25 December 2013, Volume 33 Issue 12

    

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  Gallbladder Cancer Cell Apoptosis and Up-regulated Expression of Caspase-3 Induced by Interference of Targeting MiRNA on Bmi-1

  WEI Dong, ZOU Hao, WANG Lin, WANG Wen-ju, Luo Zhi-ling, ZHANG Xiao-wen

  China Biotechnology. 2013, 33(12): 1-8.

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  Objective: Through targeted inhibition of oncogene Bmi-1 expression, to observe the induction of apoptosis and the effect of up-regulated Caspase-3 protein expression and to explore the mechanism of its formation in gallbladder cancer. Methods: The previously successfully constructed miRNA-Bmi-1 recombinant plasmid were transfected into GBC-SD cells and were divided into four groups of miRNABmi-1, miRNAScramble, Lipofectamine, GBC-SD. 48h after transfection, the mRNA and protein expression levels of Bmi-1 were measured by RT-PCR or Western blot, the cell growth was observed under inverted microscope; The apoptosis rate, cell cycle distribution and Caspase-3 protein levels were determined using flow cytometry. Results: RT-PCR and Western blot showed that Bmi-1 mRNA and protein expression of the gallbladder cancer cell in miRNA Bmi-1 group were significantly lower than those in the control groups (P<0.05). The inverted microscope showed a significantly higher cell death rate in the miRNA Bmi-1 group compared with the control groups; Cell cycle analysis showed that, in the miRNA Bmi-1 group, the cells were restrained at G0/G1 phase (72.20%±1.71), the cell number in G2/M and S phase decreased (18.30%±7.21, 9.50%±6.01) while apoptotic index increased (49.83%±5.19). Caspase-3 protein expression was increased (30.37%±8.10). Compared with the control groups, there was a significant difference (P<0.05). Conclusions: Targeting and silencing Bmi-1 can downregulate Bmi-1 mRNA and protein expression and induce the apoptosis in the gallbladder cancer cell, probably via the mechanism of restraining the early gallbladder cancer cell cycle at G0/G1 phase and up-regulating Caspase-3 protein expression. Bmi-1 may be involved in regulation of mitochondrial-dependent apoptotic pathway.
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  The Role of Canonical Wnt Signaling Pathway in the Lung Epithelial Cells against Mycobacteria Infection

  SHI Juan, YANG Jia-li, MA Ling-jie, BAO Shao-wen, MA Yan, CHENG Long, MA Chun-yan, LI Yong, LIU Xiao-ming

  China Biotechnology. 2013, 33(12): 9-14.

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  Purpose: An increasing number of studies has demonstrated that the epithelial cells of lung play a crucial role in the immunity against infections. In addition to the regulatory roles in the organogenesis and the maintenance of cell homeostasis, the canonical Wnt signaling pathway has recently been revealed to be involved in the regulation of immune responses. The aim is to investigate the potential roles of canonical Wnt signaling in the regulation of lung epithelial cells against Mycobacterium tuberculosis infection, using lung epithelial A549 cells. Methods: The A549 cells was transfected with a Wnt signaling reporter plasmid BAT flash, followed by the infection of Mycobacterium bovis vaccine strain Bacillus Calmette-Guerin (BCG). The alteration of Wnt signaling was then ascertained by measuring luciferase activity using a dual-luciferase assay, and the changes of the protein profiles of main Wnt signaling molecules and pro-inflammatory factors of the cells were determined by an immunoblotting assay or an Enzyme-linked immunosorbent assay (ELISA). Results: An inhibition of Wnt signaling luciferase activity was observed following the BCG stimulation, suggesting a negative regulation of Wnt signaling in the epithelial cells against BCG infection. Such finding was further supported by the results of immunoblotting assay and ELISA, in which an up-regulation of inhibitory proteins of Wnt signaling GSK3β(Glycogen Synthase Kinase 3 beta) and Axin2 (Axis inhibition protein) with an increased concentration of phosphorylated β-catenin, was found in the cytosol fraction of cells; and a down-regulation of Wnt signaling effector, active β-catenin and its down-stream transcription factors TCF4 (T cell factor-4) and Lef-1 (lymphoid enhancer-binding factor-1) was determined in the nuclear proteins. More importantly, overexpression of β-catenin showed a down-regulated expression of pro-inflammatory factors of interleukin-6 (IL-6), nuclear factor-kappa B (NF-κB), and the adaptor of Toll-like receptor (TLR) signaling pathway, myeloid differentiation primary response gene 88 (MyD88) and its downstream signaling TNF-receptor associated factor 6 (TRAF6), but no effect on the expression of Tumor necrosis factor-alpha (TNF-α). These data indicated that the Wnt signaling might repress the immune responses through a TLR signaling pathway in lung epithelial cells in response to BCG. Conclusion: The lung epithelial cells may able to alleviate an immune response against Mycobacteria infection through a mechanism of down-regulation of canonical Wnt signaling pathway, subsequently repress the MyD88/TRAF6/NF-κB pathway and protect the host cells from immune injury.
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  Prokaryotic Expression and Activity Detection of Ad5/Ad35 Fiber Gene

  ZHANG Wen-feng, ZHANG Qiong-yu, BO Hua-ben, SHAO Hong-wei, LI Xiao-cheng, WANG Teng, HUANG Shu-lin

  China Biotechnology. 2013, 33(12): 15-20.

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  Aim: To construct the prokaryotic expression vector for Ad5 fiber and Ad35 fiber genes, purify proteins and study the activity. Methods: Recombinant plasmid pET28a-Ad5 fiber and pET28a-Ad35 fiber were transformed into E.coli BL21(DE3) and the proteins expression were induced with IPTG. The expressed proteins Ad5 fiber and Ad35 fiber were purified by Ni²⁺ affinity chromatography and identified by SDS-PAGE. The bioactivity of proteins Ad5 fiber and Ad35 fiber were certificated by competitive binding method. Results: The proteins Ad5 fiber and Ad35 fiber can be detected in the supernatant. The proteins Ad5 fiber specifically inhibited the infectivity of the Ad5 virus in a dose-dependent manner, while had no effect on the Ad5F35-mediated gene transfer. The proteins Ad35 fiber blocked the Ad5F35-mediated gene transfer in a dose-dependent manner and had no effect on the infectivity of the Ad5 virus in competition experiments. Conclusion: Recombinant vectors Ad5 fiber and Ad35 fiber were constructed successfully. The proteins Ad5 fiber and Ad35 fiber could specifically bind with different receptors. The study will lay a foundation for the investigation on the mechanism of Ad5F35 infection.
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  Characterization of GshF Expressed in Escherichia coli

  CHEN Yong-lu, WU Mian-bin, LIN Jian-ping, YANG Li-rong, CEN Pei-lin

  China Biotechnology. 2013, 33(12): 21-28.

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  Glutathione, an important active tripeptide, is the most common non-protein thiol compound involved in cell activity. It has a wide range of applications in the pharmaceutical, food, cosmetics and other industries because of its antioxidant effect. And improving glutathione production via biosynthesis is the main method and has always been a focus. The bifunctional enzyme GshF discorvered recently can catalyze the two-step reaction of glutathione biosynthesis simultaneously and is expected to improve the catalytic efficiency. However, the properties of the enzyme have not been sufficiently studied. A bifunctional enzyme gene gshF from Streptococcus thermophilus was cloned and expressed in E. coli. GshF reached its highest activity of 44 U/L when the recombinant was induced with 0.1mmol/L IPTG at 30℃ after being cultivated for 4 hours at 37℃. The related enzymatic properties of GshF were achieved after such purification steps as cell lysis, centrifugation and affinity chromatography. The optimum pH and temperature was 8.0 and 37℃ respectively. The stabilities of GshF under its optimum pH and temperature were also studied. The final maximum yield of glutathione was 2.11g/L by enzymatic synthesis of recombinant bacteria. It is of important reference on the application of the enzyme in the biosynthesis of glutathione.
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  Expression, Purification and Biological Activity of Arginine Mutants of Bacillus subtilis RecQ Helicase

  WU Hai-li, ZHANG San-jun, DU Bing, QIAN Min, REN Hua

  China Biotechnology. 2013, 33(12): 29-34.

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  RecQ helicases are one of the most important macromolecules in the process of molecular metabolism. They play essential roles in maintaining the stability of the genetic materials in cells. The arginine residues of Bacillus subtilis RecQ play important roles in ATP hydrolyzation and binding activities. The DNA corresponding to the coding sequence of the Bacillus subtilis RecQ helicase gene was amplified by PCR from the chromosome DNA of Bacillus subtilis 168, the sequence size is about 1.5kb. The arginine residues (arg319 and arg322) of Bacillus subtilis RecQ were mutated separately or simultaneously to alanine residues by overlapping PCR method, then wild type and mutants were subcloned into the expression vector pET24a(+).The recombinant proteins were induced to express in E.coli BL21(DE3)with IPTG. All of the proteins obtained in vitro were with above 90% purity and good solubility, and then the ATP hydrolysis of wild type and mutants were detected. The results showed that Bacillus subtilis RecQ and mutants had DNA-dependent ATPase activity in concentration-dependent manner. However, the ATP hydrolysis activities of mutants were significantly reduced compared to the wild RecQ. The consequence state that the two arginine residues took important part in interacting RecQ helicaese with ATP. These results are helpful to study the structures and functions of other members of the RecQ family helicases.
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  Comparative Genomic Analysis of Lactobacillus plantarum

  CHEN Chen, REN Jing, ZHOU Fang-fang, LIU Zhen-min, GUO Ben-heng

  China Biotechnology. 2013, 33(12): 35-44.

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  As an important species of Lactic acid bacteria, Lactobacillus plantarum is closely related to the human life with various probiotic properties. With the advances of DNA-sequencing, genome determination for many strains of L. plantarum becomes available. Comparative analysis of several genomes of L. plantarum was performed based on the genome of L. plantarum ST-Ⅲ. The genes for plantaricin biosynthesis, sugar metabolism, proteolytic system and extracellular polysaccharide biosynthesis were analyzed specifically, providing advices for the research and application of this important species.
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  Effects of Sucrose Feeding Rate and pH Control on Nisin Production in Fermentor

  WANG Rui, DU Chao, ZUO Fang-lei, CHEN Shang-wu

  China Biotechnology. 2013, 33(12): 45-50.

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  Effects of the pH control and sucrose feeding rate during the fermentation period on the fermentation of Lactococcus lactis and the titer of nisin were mainly studied. Through the comparison with the nisin produced by Lactococcus lactis which not adjust the fermentation condition pH, it is found that titer of nisin yeild 1.2 times higher than that nisin without pH asjustment which adjust the fermentation condition pH to the optimum value of 6.8. The sucrose feeding rate was designed at 2 and 4 g/L·h, respectively, nisin titer increase about 50.5% by constant addition of sucrose at a feeding rate of 4 g/L·h.
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  Establishment and Application of a System for Drug Screening Targeting MERS-CoV Main Proteinase

  ZHANG Ning, PAN Li, NIU Guo-jun, WANG Wen-ming, ZHOU Hong-gang, YANG Cheng

  China Biotechnology. 2013, 33(12): 51-56.

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  Objective: Targeting MERS-CoV main proteinase NSP5, a new system for drug screening was established for inhibitors screening by using fluorescence resonance energy transfer (FRET). Methods: Target protein was obtained using prokaryotic expression system and technologies of gene recombination, protein-expression and purification. Proteinase activity of target protein was examined by FRET assays. Finally, a system for drug screening was established and optimized before abundant compounds screening. Results: Using the drug screening system, eight compounds with high inhibition ratio were selected. Notably, the IC₅₀ of MDCCCL002330 is minimal, namely 0.43μmol/L. Conclusion: The established system targeting MERS-CoV main proteinase NSP5 is suitable for inhibitors screening, which can be used to promote research and development of lead compounds.
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  Preliminary Establishment of High Resolution Melting Analysis for Detecting IDH2 Gene Mutations in Gliomas

  WANG Yin-hui, ZHANG Hai-yan, YANG Chuan-hong, ZHANG Wei, LAI Huang-wen, CHEN Xiao-dong, WANG Jie

  China Biotechnology. 2013, 33(12): 57-63.

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  Isocitrate dehydrogenase2(IDH2) mutations have recently been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas, as well as secondary glioblastomas. Since the mutations have been associated with better clinical outcome, they have shown diagnostic and prognostic value. It was performed with optimizing conditions to preliminary establish a high resolution melting for the detecting of IDH2 mutations in routine formalin-fixed tissues of brain biopsies. IDH2 mutations in paraffin-embedded tissues of gliomas were detected by HRM and direct sequencing, estimating the stability and sensitivity of reaction system and reaction conditions. The optimal annealing temperature was 52.5 ℃. The optimization of 20 μl-HRM reaction system including 0.6 μmol/L primer, 45 ng DNA template,2.75 mmol/L Mg²⁺, was established by response surface Design. The results were consistent with the direct sequencing but have a higher sensitivity. It showed that HRM was simple, specific, accurate and reliable method. It would provide the reference for the clinical application of detection IDH2 mutations.
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  Establishment of Homogeneous Time-resolved Fluorescence (HTRF) Method for Measurement of Monoclonal Antibody Concentration

  DENG Chun-pin, QIN Yu-min, DENG Qiao-chun, WANG Bo, PENG Yu-cai

  China Biotechnology. 2013, 33(12): 64-68.

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  A rapid and simple antibody analysis method based on HTRF technology was established to measure concentration of recombinant monoclonal antibody in both cell culture medium and purified antibody solution. APC-labeled antigen,antibody samples or controls, and Eu-labeled anti-human IgG are added into 96 well plate (Black), incubate for some time, followed by fluorescence reading with microplate reader. Standard curve was generated by 4-PL model, antibody concentration in tested samples was calculated accordingly by software. The method qualification results showed that the method is antigen-antibody specific, with accuracy close to 98.9%, precision close to 4.3%, and linearity range 25～1000 ng/ml. This method can be used for cell line screening, bio-process monitoring, and quality control during antibody drug development.

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  Simultaneous Over-expressions of AOD1 and HPS-PSI in Chloroplasts Creates a Novel Photosynthetic CH₃OH-assimilation Pathway and Enhances Its Ability to Assimilate CH₃OH

  LIU Lei, SUN Zhen, SONG Zhong-bang, XIAO Su-qin, CHEN Li-mei

  China Biotechnology. 2013, 33(12): 69-78.

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  3-hexulose-6-phosphate synthase (HPS) and 6-phosphate-3-hexuloisomerase (PHI) are two key enzymes in a HCHO-assimilation pathway in methylotroph. It has demonstrated that overexpression of HPS/PHI fusion protein in chloroplasts of geranium installs a photosynthetic HCHO-assimilation pathway and thereby enhanced the ability of transgenic plants to assimilate and detoxify formaldehyde. Alcohol oxidase (AOD1) is the first key enzyme in the methanol metabolic pathway in methylotrophic yeasts. AOD catalyzes oxidation of methanol to formaldehyde. AOD1 or/and HPS-PHI were over-expressed in chloroplasts of tobacco to generate AOD1 (AO), HPS-PHI (AB) over-expression line as well as AOD1 and HPS-PHI (AA) simultaneous over-expression line. The transgenic and wild-type tobacco (WT) was treated with 2 mmol/L and 6 mmol/L ¹³CH₃OH. ¹³C-NMR analysis showed that the accumulation of H¹³COOH was maximum in AO trangenic plants. There is no significant difference in the generation of [U-¹³C] glucose (Gluc) and [U-¹³C] fructose (Fruc) between AO and WT lines. However, the production of [U-¹³C] glucose (Gluc) and [U-¹³C] fructose (Fruc) in AA and AB lines was significantly higher than that in AO and WT lines. Moreover, the formation of the two metabolites in AA line was higher than in AB line. These differences were more significant in 6mmol/L ¹³CH₃OH treated-plants than in 2 mmol/L ¹³CH₃OH-treated plants. These results suggested that overexpression of AOD1 in chloroplasts enhanced the ability of tobacco to oxidize methanol to formaldehyde, while simultaneous overexpression of AOD1 and HPS-PHI could successfully created a photosynthetic CH₃OH-assimilation pathway, thus enhanced ability of tobacco to assimilate CH₃OH as sugars.
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  The Stability Reconstruction of β-mannanase with N-glycosylation Modification

  XIE Chun-fang, LI Yu-feng, LIU Da-ling, YAO Dong-sheng

  China Biotechnology. 2013, 33(12): 79-85.

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  N-glycosylation is one of the most important posttranslational modification of proteins in eukaryotes. A rational strategy to introduce N-glycosylation site was proposed to Armillariella tabescens beta mannose Man47.Then g-123 mutant with EAS(enhanced aromatic sequence) sequence was built through the molecular docking, secondary structure analysis and feasibility analysis of glycosylation. The sequence of g-123 mutant was inserted into SMD1168 with the yeast α-mating factor, then transformed it into Pichia by electroporation to obtain the recombinants. Finally the thermal stability, acid and alkali stability, pepsin-resistance and trypsin-resistance of g-123 and wild type were analyzed. The results showed that compared with wild type, the thermal stability, acid and alkali stability, protease resistance of the mutant g-123 with glycosylation was improved.
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  Modification of 5’UTR Sequences of pPIC9K Increases Expression of Antimicrobial Peptide PR39

  MING Fei-ping, YANG Jun, ZHU Jin-mei, KUANG Zhe-shi, LI Hua-zhou, XIA Feng-geng, YE Ming-qiang, WANG Hou-guang, ZHAO Xiang-jie, HUANG Zhi-feng, MA Miao-peng, SHI Ju-qing, CAI Hai-ming, ZHANG Ling-hua

  China Biotechnology. 2013, 33(12): 86-91.

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  Objective: To express antibacterial peptide PR39 in Pichia pastoris and determine the activity of product.Methods: Four oligonucleotide fragments were synthesized according to the codon bias of Pichia pastoris,and complete coding sequence was obtained by overlapping PCR, then the sequence was cloned to expression vector pPIC9K. The sequence by Kex2 to the first nucleotide of PR39 was knocked out on the recombinant plasmid pPIC9K-PR39, then the sequence of GGATCCAA in 5'UTR was also deleted by PCR-Restriction Enzyme ligation method, finally a new expression vector named pPIC9K-PR39-D-E was obtained. The vector pPIC9K-PR39-D-E was transformed to Pichia pastoris SMD1168, and clones were identified by PCR and than screened with G418 for expression under induction of 0.5% methanol. The expressed product was identified by Tricine-SDS-PAGE, and determined for antibacterial activity by agarose diffusion test,purified by reversed phase chromatography and ion-exchange column chromatography. Results: PR39 with 4.7kDa and reached 175.6mg/L after purification. It showed antibacterial activity to E.coli DH5α. Conclusion: PR39 was successfully expressed in Pichia pastoris, which laid a foundation of further study on antibacterial peptide.
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  Study on Moderate Wall-breaking Preparation Bioprotein of Yeast Source

  WANG Bin, HUANG Li-na, QIU Li-juan

  China Biotechnology. 2013, 33(12): 92-96.

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  It is mainly focused on the yeast cell wall impeding nutrients from efficient digestion and absorption by the body. We investigated the cell wall breaking process, protein delivery rate, active substance preservation, product flavor and animal breeding effect by utilization of a technique consisting of three sections of variable temperature autolysis plus enzyme digestion. Consequently a moderate wall-breaking method with improved yeast protein availability, better nutrient preservation and flavor, and easy industrialization has been established. This will provide a large number of high-quality proteins for human health and animal breeding.
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  Optimization of Fermentation Process for Chlamydospores of Trichoderma asperellum by Response Surface Methodology

  WU Wei-ping, CHEN Jie, LI Ya-qian, CHEN Li-jie, DUAN Yu-xi

  China Biotechnology. 2013, 33(12): 97-104.

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  The response surface methodology (Box-Benhnken Design) was applied to investigate the four major factors (the concentration of soybean cake powder and maize powder used in medium, the volume of glycerol addition added to the medium, loading volume of liquid medium, initial pH value of medium) that have importent effect on the production of chlamydospores of Trichoderma asperellum ZJSX5003. By variance analysis on the experiment results, the influences of the the four factors and their interactions on the production of chlamydospores was investigated. By performing regression analysis and establishing regression equation based on statistics, the optimal fermentation condition was obtained, that as follow:the concentration of soybean cake powder and maize powder used in medium 33.25g /L, glycerol 8.86ml/L, each 500ml bottles contains 99.35ml liquid medium, initial pH 3.26. Under the optimal conditions,the yield of chlamydospore is expected to be 9.56×10⁷ spores/ml, theoretically. The practical yield of chlamydospores approached 9.84×10⁷spores/ml, reached 97.07% of the expected value, increased by 69.07% compared with pre-optimization. In order to test its applicability, used a 10L fermentation tank under the optimal condition, the yield of chlamydospore reached up to 1.75×10⁸spores/ml,which is better than shake flask fermentation. Further researches of developing microbial inoculum of Trichoderma chlamydospore could be conducted base on this research.

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  The Clinical Research Progress for Oncolytic Adenovirus Targeting Cancer Therapy

  ZHOU Li, HE Wan-wan, ZHU Zhen-nan, YANG Yuan-qin, ZHAO Hong-fang, MA Bu-yun, WANG Yi-gang

  China Biotechnology. 2013, 33(12): 105-113.

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  Adenovirus has been discovered and successfully isolated since the 1950s,then biologists conduct extensive researches on oncolytic adenovirus.Compared to other viruses,oncolytic adenovirus could infect tumor cells specifically and accomplish an infection-replication cycle in tumor cells,which can specifically kill and destroy cancer cells,but not damage normal cells or tissues.It has become one of the most promising antineoplastic medicines. However,at the process of clinical gene therapy, adenovirus vectors present the problems that lacking tissues or cells specificity,low genetic transfection efficiency of targeting cells and the lack of certain safety.Therefore,it is rather crucial to improve the targeting ability of recombinant adenovirus for cancer gene therapy.According to relevant studies, a variety of oncolytic adenoviruses as vectors of cancer gene for therapy have entered into related clinical trials involving different kinds of cancers,which shows admirable clinical application. It is mainly to focus on the oncolytic adenovirus types, cancer targeting strategies and clinical studies.
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  Research Progress on Prevention and Controlling of Bacterial Contamination in Biomass Fermentation

  LIU Hua-qing, LI Hao

  China Biotechnology. 2013, 33(12): 114-120.

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  Biomass energy has many advantages, and it is considered to be environmentally-friendly, low cost, renewable and sustainable. However, fermentation of biomass energy is still facing some issues, such as immature technology of non-food raw materials, hypertonic cells, high temperature, product stress, as well as bacterial contamination. Bacterial contamination is a long-standing problem that cannot be completely solved in the biomass fermentation industry. Bacteria usually inhibit fermentation by affecting the fermenting environment and competing substrate and survival environment, etc. If it cannot be effectively controlled, it will seriously affect the production, which may lead to huge economic losses. Traditional way to prevention and control bacterial contamination is adding antibiotics. But the resistance problems have become increasingly prominent, so some new bacteria-prevention methods are paid more and more attention. The recent progress on the causes of bacterial contamination in bio-ethanol and other biomass fermentation, the mechanism of bacterial contamination inhibiting biomass energy fermentation, and the research progress on how to prevent and control bacterial contamination were discussed.
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  The Fermentation Advantage of Kluyveromyces lactis and Its Application in the Field of Food Enzyme

  WANG Yu-hai, ZHANG Hui-bin, YANG Hui-min, MENG Qing-qing, WANG Feng-huan

  China Biotechnology. 2013, 33(12): 121-126.

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  Kluyveromyces lactis is the yeast that can use lactic acid as its sole carbon and energy source, with the extremely simple nutritional requirements, vigorous growth, large biomass growth,temperature adaptation to a wide range, good secreting function of protein, does not produce endotoxin, human security and other advantages. It has also shown great potential as a host system to express food enzyme. From biological characteristics, strain selection, fermentation process optimization, analysis of the effect of expression, modification of the heterologous protein these many aspects to elaborate the fermentation characteristics of Kluyveromyces lactis, and pointed out that it has a unique fermentation advantage. Providing a brief overview of Kluyveromyces lactis used in the field of food enzyme.
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  Problems and Solutions in the Drafting of Biotechnological Patents

  WU Xue-mei, QU Kai

  China Biotechnology. 2013, 33(12): 127-132.

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  The Biotechnological patent application is somewhat special in technology exertion, law and regulations and social ethics. The common problems and solutions in this field were analyzed, including subject matters for which no patent right shall be granted, insufficient disclosure of the invention, unsuitably generalization of claims and so on. It will provide enough help for patent drafting in this field.
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  Comparison of Patent Examination Between China and Japan Concerning Novelty and Inventiveness of Protein Products

  SUN Qian, XU Li, ZHOU Mao-xin, ZHONG Hui

  China Biotechnology. 2013, 33(12): 133-136.

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  In fields of bio-engineering development and patent examinations, China and Japan have certain similarities. Specific to protein products, their criteria for inventiveness are basically the same. Meanwhile, Japan has the additional provision besides the case in China's "Guidelines for Patent Examination" in protein's novelty, as proteins differ from known ones only in terms of oligosaccharide chain is novel. China's invalidation case of No. 15463 and Japan's Heisei 14 No.505 precedent embody the examination standards, which have guiding significance in future's judgments.