GM-CSF GENE OR B7-1 GENE MODIFIED MURINE EL-4 CELLS VACCINE

Objective: To study the vaccine potency of gene-modified tumor cells. Methods: The EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene or B7-1 gene. The effect of gene transduction on antitumor immunity was investigated. Results: Flow cytometry analysis showed that expression of their surface marker between wild-type EL-4 cells and gene transduced tumor cells was the same except for CD80 positive in B7-1 gene transduced cells. GM-CSF gene or B7-1 gene transduced EL-4 cells resulted in remarkable loss of tumorigenicity in syngenetic mice. The systemic protective immunity was induced against the challenge with EL-4/wt cells. Therapeutic vaccine with EL-4/GM-CSF or EL/7-1 cells could retard the growth of established early-stage EL-4/wt tumor significantly, but not retard the growth of late-stage EL-4/wt tumor. Irradiated GM-CSF gene transduced EL-4 cells showed strong vaccine effect against EL-4 cell challenge, but irradiated B7-1 gene transduced EL-4 cells showed weak vaccine effect. Remarkable cooperative antitumor effect against EL-4 cell challenge was observed when both irradiated EL-4/GM-CSF and EL-4/B7-1 were inoculated together. Conclusion: GM-CSF gene or B7-1 gene transduced BL-4 cells can be used as a good tumor vaccine. The combination of the two kinds of vaccine may have potential application value in human cancer treatment.

Research Progress of Medicinal Secondary Metabolites and Gene Cloning of Dendrobium officinale

Dendrobium officinale is one of the most precious medicinal plants in China. Its main medicinal ingredients are its secondary metabolites. However,it has the characteristics of limited sources,low active ingredient content and high cost,limiting the use of D. officinale.Studying the network structure and rate-limiting steps of secondary metabolites of medicinal components of D. officinale,analyzing the secondary metabolic synthesis process,mastering the production rules of its medicinal components and carrying out gene cloning or biosynthesis,etc.are of great significance for the rational development and utilization of D. officinale resources. This paper briefly reviews the progress of the research on the secondary metabolites of D. officinale,including the detection and identification of metabolites and the identification and cloning of key metabolic enzymes.

Identification of Senescence-associated Protein DpXTH1 and its Gene Cloning in Dahlia Petals

[Objective] This study aimed to explore the molecular mechanism of senescence in ethylene-insensitive flowers. [Method] The dahlia petals were used as matedal, and the senescence-associated proteins were isolated and identified using two-dimensional electrophoresis, mass spectrometry and an encoding gene was cloned using molecular biology techniques. [Result] In the two-dimensional elec- trophorogram of proteins from dahlia petals at building color, full flowering and flow- er senescence pedods, a total of 44 protein spots with differences in expression level more than two times were detected. From the 44 protein spots, xyloglucan （XTHs）, a senescence-associated protein, was iso- lated and identified and its expression level was increased continuously with the senescence process of dahlia petals. By using the total RNA of dahlia petals as matedal and a pair of degenerate pdmers, the cDNA sequence of XTH gene was cloned by RT-PCR. The encoding region of XTH gene has a full length of 882 bp, encoding 293 amino acid residues, and is named as DpXTH1 （Accession number： HM053613.1）. The cluster analysis showed that the amino acid sequence of DpXTH1 has high homology with those of XTHs in other plants. [Conclusion] The isolated and identified DpXTH1 from dahlia petals belonged to the XTH family in plants, and its biological function was associated with the senescence process and regulation of dahlia petals.

A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U （mg protein）-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity （pH 5,0-6.0）. The enzyme was activated by Na＋, Mg2＋, Ca2＋, K＋, Fe2＋ and Cu2＋, however, it was inhibited by Fe3＋, Ba2＋, Zn2＋, Mn2＋ and Ag＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol L-1 min-1 （mg protein）-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose （CMC） after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading flame of 954bp （EU978473）. The protein deduced contained the conserved domain of cellulase superfamily （glucosyl hydrolase family 5）. The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

The Construction of the Probe for Amylase n Gene Cloning from Bacillus halodurans Strain 38C1-1

Primers and probes were established according to the sequences of the alpha-amylase genes of Bacillus. halodurans C-125, Therrnus sp. IM6501, B. stearothermophilus ET-1, and B, acidopullulytics. Primers were designed and a 0.2 kb DNA fragment was amplified, the fragment was successfully used for the detection of the amylase Ⅱ gene in a 2 842 bp region from Bacillus halodurans strain 38C1-1.

huIL-6-GM-CSF(9-127)融合蛋白的表达及活性测定

目的 构建及表达具有huIL 6和huGM CSF双重生物学活性的huIL 6 GM CSF(9- 12 7)融合蛋白分子。方法 应用PCR技术对huIL 6和huGM CSF的基因分别加以改造 ,同时在二者之间加上连接肽序列 (G G S G S) 3 ,克隆PCR产物 ,并构建成pBV2 2 0 IL 6 GM CSF(9- 12 7)表达质粒 ,将表达质粒导入E .coliDH5α中 ,诱导表达融合蛋白。通过QSepharoseH .P .离子交换柱和SephacrylS 2 0 0分子筛柱二步柱纯化以获取目的蛋白。使用MTT法测量其huIL 6和huGM CSF生物学活性。结果 pUC18 IL 6 GM CSF(9- 12 7)的序列与理论设计完全一致 ,表达质粒在E .coliDH5α中得到高效表达 ,表达的融合蛋白占总蛋白含量的 30 %以上 ,表达产物以包涵体的形式存在。通过QSepharoseH .P .离子交换柱和SephacrylS 2 0 0分子筛柱二步柱纯化及复性后获得目的蛋白 ,其纯度达到 96 %以上。融合蛋白具有huIL 6和huGM CSF的双重生物学活性 ,其促进huIL 6依赖细胞株B9和huGM CSF依赖细胞株TF 1增殖的比活性分别为 2 .86× 10 7U/mg和 3.33× 10 8U/mg。结论 获得了具有较高纯度和双重生物学活性的huIL 6 GM CSF(9- 12 7)

huGM-CSF(9-127)-IL-6(29-184)融合蛋白基因的构建及表达

目的构建及表达具有huGM-CSF和huIL-6双重生物学活性的huGM-CSF(9-127)-IL-6(29-184)融合蛋白分子。方法应用PCR技术对huGM-CSF和huIL-6的基因分别加以改造,同时在二者之间加上连接肽序列(G-G-S-G-S)3,克隆PCR产物,并构建成pBV220-GM-CSF(9-127)-IL-6(29-184)表达质粒,表达质粒导入E.coliDH5α中诱导表达。通过QSepharoseH.P.离子交换柱和SephacrylS-200分子筛柱二步柱纯化以获取目的蛋白。使用huGM-CSF依赖细胞株TF1和huIL-6依赖细胞株B9通过MTT法测量融合蛋白的生物学活性。结果pUC18-GM-CSF(9-127)-IL-6(29-184)的测序结果表明,其序列与理论设计完全一致,表达质粒在E.coliDH5α中得到高效表达,表达的融合蛋白占总蛋白含量的25%以上,表达产物以包涵体的形式存在,通过QSepharoseH.P.离子交换柱和SephacrylS-200分子筛柱二步柱纯化及复性后获得目的蛋白,其纯度达到95%以上。融合蛋白具有huGM-CSF和huIL-6的双重生物学活性,其促进huGM-CSF依赖细胞株TF-1和huIL-6依赖细胞株B9增殖的比活性分别为1.08×108U/mg和1.95×107U/mg。结论获得了具有较高纯度和双重生物学活性的GM-CSF(9-127)-IL-6(29-184)融合蛋白。

犬GM-CSF基因的克隆、序列分析及蛋白结构预测

为了研究犬粒细胞/巨噬细胞集落刺激因子(GM-CSF)的作用,试验根据GenBank上发表的犬(Canis familiaris)GM-CSF cDNA基因序列设计1对引物,采用RT-PCR技术,以ConA刺激的犬外周血淋巴细胞为材料,从总RNA中扩增出犬GM-CSF基因。结果表明:扩增片段长为435 bp,编码144个氨基酸;生物软件分析结果表明,该序列与猕猴的亲源性更近,与已知人、马、牛、野猪、豹猫、鸡等序列的同源性较低。

鸡粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因的克隆及生物信息学分析

为了解GM-CSF的基因及蛋白质特性,本研究以pUC-GM-CSF为模板,根据GenBank中登录的GM-CSF序列设计引物扩增GM-CSF基因,随后将其克隆至pMD18-T载体,并对鉴定正确的重组质粒pT-GM-CSF进行测序分析,然后对GM-CSF基因进行同源性分析、ORF分析、氨基酸序列分析、疏水性分析、磷酸化位点预测、亚细胞定位分析及二级结构预测。结果表明:该基因ORF的全部长度为453bp,能够编码150个氨基酸,并且在GM-CSF基因所编码的蛋白质中有4个丝氨酸和3个苏氨酸可能成为蛋白激酶磷酸化的位点。同时,GM-CSF蛋白的最大疏水性为2.267,且有8.91%的可能位于细胞外,有0.8%的可能位于细胞壁,有0.24%的可能位于细胞质。同时,该蛋白质的二级结构主要由无规卷曲、α螺旋和延伸链构成。本研究通过对GM-CSF基因进行生物信息学分析,为探究其免疫增强机制奠定基础。

猪粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因的克隆和序列分析及其基因表达

采取三元杂交猪的外周血,提取单核细胞总RNA,采用RT-PCR克隆猪粒细胞-巨噬细胞集落刺激因子(GM-CSF)cDNA(GenBank登录号为DQ108393)。以pcDNA3.1(-)为质粒载体,构建猪GM-CSF基因真核表达质粒pGM-CSF,对克隆片段进行测序,并进行不同物种间同源性的比较。将重组质粒转染COS-7细胞,Western blotting鉴定表达蛋白的特异性。序列分析表明,GM-CSF的cDNA开放阅读框为435 bp,编码144个氨基酸和大部分信号肽,与用作PCR引物设计的参考序列比较,其核苷酸的同源性为97.5%,氨基酸的同源性为95.1%,同绵羊、人、牛、小鼠和犬的GM-CSF序列比较,其核苷酸的同源性分别为86.2%、80.5%、81.7%、69.7%和81.8%,氨基酸的同源性分别为78.5%、71.3%、70.3%、55.9%和71.5%。Western blotting证实目的基因可以在真核细胞中特异性的表达GM-CSF蛋白。以上结果表明,猪的GM-CSF基因被成功克隆,它存在着种属特异性。这为进一步研究该基因的生物学作用特别是利用GM-CSF增强DNA疫苗的免疫效果奠定了基础。

人IL-3、GM-CSF和Hly基因的cDNA克隆及测序

试验利用PCR技术扩增并获得了人IL-3、GM-CSF和人溶菌酶(human lysozyme,Hly)基因组基因编码区序列,人IL-3和GM-CSF基因PCR产物与pMD19-T连接,Hly基因PCR产物同pEASY-Blunt载体连接,转化大肠杆菌JM109感受态细胞,蓝白斑筛选后经PCR及酶切鉴定,选取测序正确的克隆连接到pIRESneo载体中构建相关真核表达载体,分别用这些基因的表达载体转染PK15细胞,48 h后提取转染细胞的总RNA,并采用RT-PCR方法获得了人IL-3、GM-CSF和Hly基因的cDNA,结果发现,所获得的cDNA序列同NCBI登录的序列完全同源。

重组hGM-CSF乳酸链球菌的构建及初步验证

目的运用基因克隆技术将自行合成的人粒-巨噬细胞集落刺激因子(hGM-CSF)转化于乳酸链球菌,并筛选获得高表达重组hGM-CSF的乳酸链球菌稳定株。方法将经过优化适合在乳链菌表达的hGM-CSF基因克隆于含有P59启动子、USP45蛋白信号肽、终止密码子的pNBC1000载体,得到重组质粒pNCSF,然后将pNCSF进一步克隆于穿梭载体pTR1001c,获得乳链菌表达载体pTRCSF,通过电穿孔转化于乳链菌,并通过聚丙烯酰胺凝胶蛋白电泳验证hGM-CSF的表达。结果获得了重组质粒pNCSF并经酶切鉴定和测序证实,酶切鉴定显示构建乳链菌表达载体pTRCSF及重组hGM-CSF乳链菌成功,蛋白电泳初步验证获得了高表达重组hGM-CSF的乳链菌株LL-CSF。结论运用基因克隆技术成功构建了重组乳酸链球菌LL-CSF,为进一步研究重组hGM-CSF的生物学特性及评价其潜在临床应用价值打下了基础。

Cost-Effective Method of Gene Synthesis by Sequencing from Microchip-Derived Oligos for Droplet Cloning

Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthesis has been increasing for the past few decades, yet available methods remain expensive. A solution to this problem involves microchip-derived oligonucleotides (oligos), an oligo pool with a substantial number of oligo fragments. Microchips have been proposed as a tool for gene synthesis, but this approach has been criticized for its high error rate during sequencing. This study tests a possible cost-effective method for gene synthesis utilizing fragment assembly and golden gate assembly, which can be employed for quicker manufacturing and efficient execution of genes in the near future. The droplet method was tested in two trials to determine the viability of the method through the accuracy of the oligos sequenced. A preliminary research experiment was performed to determine the efficacy of oligo lengths ranging from two to four overlapping oligos through Gibson assembly. Of the three oligo lengths tested, only two fragment oligos were correctly sequenced. Two fragment oligos were used for the second experiment, which determined the efficacy of the droplet method in reducing gene synthesis cost and speed. The first trial utilized a high-fidelity polymerase and resulted in 3% correctly sequenced oligos, so the second trial utilized a non-high-fidelity polymerase, resulting in 8% correctly sequenced oligos. After calculating, the cost of gene synthesis lowers down to 0.8 cents/base. The final calculated cost of 0.8 cents/base is significantly cheaper than other manufacturing costs of 7 - 30 cents/base. Reducing the cost of gene synthesis provides new insight into the cost-effectiveness of present technologies and protocols and has the potential to benefit the fields of bioengineering and gene therapy.

Gene Cloning and Bioinformatics Analysis of phoR Gene from Vibrio alginolyticus HY9901

PhoR is a histidine kinase in a two-component regulatory system that regulates phosphorus metabolic pathways and undertakes the key mission of information transmission in pathogenic bacteria.The full-length phoR gene was successfully cloned from the Vibrio alginolyticus HY9901 strain.A comprehensive analysis of the cloned gene was conducted using bioinformatics.Sequence analysis revealed that the total length of the phoR gene(GenBank accession No.:KJ958404.1)is 1299 bp,with the coding region containing a total of 432 amino acid residues.The phylogenetic tree of PhoR revealed that it belongs to the same subclade as V.diabolicus.The SMART program was employed for the purpose of functional domain prediction,which revealed that PhoR possesses three major functional domains:PAS(amino acids 98-166),HisKA(amino acids 205-272),and HATPase_c(amino acids 317-429).

Molecular Cloning of sodB Gene from Vibrio alginolyticus HY9901 and Its Bioinformatics Analysis

Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino acids.The predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of 4.95.Upon prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane region.The amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal site.The probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the protein.The amino acid sequence of the sodB gene of V.alginolyticus exhibited 98%-100%homology to other Vibrio species,clustering into the same subfamily with V.parahaem,indicating a relatively close relationship between them.In the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,respectively.The similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.

Molecular Cloning and Bioinformatics Analysis of msrA Gene from Vibrio alginolyticus Strain HY9901

[Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With Vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of msrA,and its bioinformatics analysis was carried out.[Results]The full length of msrA gene was 639 bp,encoding 212 amino acids,and its theoretical molecular weight was about 23729.60 Da.The protein had a stable structure,and it was hydrophobic overall.The structure of signal peptides at the N terminal of the amino acid sequence was predicted,and it was found that there was no signal peptide cleavage site and no transmembrane region.The amino acid sequence of MsrA contained multiple signal binding sites.Protein subcellular localization showed that MsrA protein was most likely located in the cytoplasm.Homology analysis showed that MsrA of V.alginolyticus had high homology with other Vibrio species,and the highest homology with V.alginolyticus.In the prediction of functional domains,MsrA had the function of methionine sulfoxide reduction.In secondary structure prediction,MsrA contained random coils at a proportion of 46.70%,which was the highest.The similarity between the tertiary structure model of MsrA and template Q87SW6.1.A was 89.15%.PTM analysis showed that MsrA protein had many PTM modification sites such as phosphorylation and glycosylation sites.[Conclusions]This study provides some reference value for further study on the role of MsrA in bacterial antioxidant stress.

乳链菌表达hGM-CSF基因的拼装及其载体pNCSF构建

目的体外拼装适合在乳酸链球菌表达的人粒-巨噬细胞集落刺激因子(granulocyte-macrophagecolonystimulatingfactor,hGM-CSF),并构建载体pNCSF。方法根据hGM-CSF的氨基酸序列及乳酸链球菌偏好的密码子,将hGM-CSF基因外显子的全长碱基序列用DNAWORKS程序设计成16条互补的寡核苷酸片段,并且分别在第1条和第16条寡核苷酸片段上分别设计PstⅠ和BamHⅠ酶切位点,将相同终浓度的寡核苷酸混合进行PCR反应,完成hGM-CSF基因拼接,得到目的基因片段,对寡核苷酸片段进行拼装和扩增,产物稀释后进一步纯化扩增,在Tag酶存在的条件下对产物两末端加T,然后TA克隆于pGEMTeasy载体中,经酶切鉴定得到阳性克隆并经测序验证。然后亚克隆于含有P59启动子、USP45蛋白信号肽的pNBC1000载体。结果通过PCR得到420左右目的DNA片段,获得了hGM-CSF和pNCSF阳性克隆,其DNA测序结果与设计序列完全一致。结论运用基因拼装及克隆技术成功拼装了hGM-CSF基因及构建了载体pNCSF,为乳酸链球菌表达重组hGM-CSF奠定了基础。

两种hIL-6/GM-CSF融合蛋白基因的构建、表达及活性比较

目的 :构建及表达具有hIL 6和hGM CSF双重生物学活性的hIL 6 /GM CSF融合蛋白分子。方法 :应用PCR技术对hIL 6和hGM CSF的基因分别加以改造 ,同时在两者之间加上连接肽序列 (G G S G S) 3 ,克隆PCR产物 ,并构建成克隆有两种融合蛋白基因的pBV2 2 0表达质粒 ,两种融合蛋白分别是 :IL 6 (1～ 184 ) GM CSF(9～ 12 7) (简称IG1)和IL 6 (2 4～ 184 ) GM CSF(9～ 12 7) (简称IG2 )。将表达质粒分别导入E .coliBL 2 1中诱导表达。通过QSepharoseHP离子交换柱和SephacrylS 2 0 0分子筛柱二步柱纯化目的蛋白。使用hIL 6依赖细胞株B9和hGM CSF依赖细胞株TF1,通过MTT比色法测定融合蛋白的生物学活性。结果 :对两种融合蛋白基因的测序结果表明 ,其序列与理论设计完全一致。表达质粒在E .coliBL 2 1中均得到高效表达 ,表达的融合蛋白均占总蛋白含量的 2 5 %以上 ,表达产物以包涵体的形式存在 ,通过QSepharoseHP离子交换柱和SephacrylS 2 0 0分子筛柱二步柱纯化及复性后 ,获得两种目的蛋白 ,其纯度均达到 95 %以上。活性测定结果表明 ,两种融合蛋白均具有较高的hIL 6和hGM CSF的双重生物学活性。结论 :获得了具有较高纯度和双重生物学活性的hIL 6 /GM

利用PCR技术构建GM-CSF/Fc_(γ2)^-融合蛋白真核表达载体

目的 对融合基因GM -CSF Fcγ2 进行定点突变 ,去除其补体结合位点 ,构建GM -CSF Fcγ2-融合蛋白真核表达载体。方法 通过设计突变引物 ,利用PCR定点突变技术扩增融合基因GM -CSF Fcγ2-。并通过T -A克隆策略 ,把突变后的融合基因GM -CSF Fcγ2-重组到真核表达载体pRc CMV2中 ,构建真核表达质粒pRc CMV2 GM -CSF Fcγ2-并经过酶切和测序确证。结果 成功对融合基因GM -CSF Fcγ2-进行了突变 ,构建了真核表达载体pRc CMV2 GM -CSF Fcγ2-。

利用PCR技术构建GM-CSF/Fc γ_2融合蛋白载体

目的：构建 Ｇ Ｍ Ｃ Ｓ Ｆ／ Ｆｃγ２ 融合蛋白载体。方法：利用 Ｐ Ｃ Ｒ技术，将人 Ｇ Ｍ Ｃ Ｓ Ｆ基因与人免疫球蛋白 Ｉｇ Ｇ２ 的 Ｆｃ 段基因联接，构建融合基因ｈ Ｇ Ｍ Ｃ Ｓ Ｆ Ｈ Ｖ２；并将此片段定向克隆到真核表达质粒ｐ Ｒｃ／ Ｃ Ｍ Ｖ２ 载体。结果：琼脂糖凝胶电泳结果显示 Ｐ Ｃ Ｒ扩增出了 １．３ ｋｂ 的融合基因片段；限制性内切酶图谱分析，成功地完成了 Ｇ Ｍ  Ｃ Ｓ Ｆ／ Ｆｃγ２ 融合蛋白载体的构建。结论：（１）经 Ｐ Ｃ Ｒ 扩增技术由质粒 Ｐ Ｃ Ｄｈ Ｇ Ｍ Ｃ Ｓ Ｆ和 Ｐ Ｓ Ｖ Ｖ Ｎ Ｐ Ｈ Ｖ２ 分别扩增到了所需的ｈ Ｇ Ｍ Ｃ Ｓ Ｆ ｃ Ｄ Ｎ Ａ 片段和 Ｉｇ Ｇ２ 从绞链区到 Ｃ Ｈ３ 的基因组 Ｄ Ｎ Ａ 片段。（２）利用 Ｐ Ｃ Ｒ介导，扩增出了融合基因片段ｈ Ｇ Ｍ Ｃ Ｓ Ｆ Ｈ Ｖ２。（３）构建了真核细胞表达载体ｐ Ｒｃ／ Ｃ Ｍ Ｖ２／ｈ Ｇ Ｍ Ｃ Ｓ Ｆ Ｈ Ｖ２ 。