Bioactivity and secretion of interleukin-18 (IL-18) generated by equine and feline IL-18 expression constructs

Interleukin 18 (IL-18) is a cytokine capable of induction of IFNgamma, granulocyte monocyte-colony stimulating factor (GM-CSF), TNFalpha and IL-1 in immunocompetent cells. Equine and feline plasmid vectors expressing pro-IL-18, mature IL-18 and IL-18 fused to a synthetic signal sequence from human IL-1beta receptor antagonist protein (ILRAP), ILRAP-IL-18, have been generated. In vitro protein expression of these constructs was compared by Western blot analysis. These data demonstrated that ILRAP-IL-18 protein was secreted readily from transfected chinese hamster ovary (CHO) cells. A simple bioassay for human IL-18 was recently described using human myelomonocytic KG-1 cells, which produce human IFNgamma in response to human IL-18 in a dose dependent manner (Konishi et al., 1997). We demonstrated bioactivity of equine and feline IL-18 protein in transfection products of CHO cells using this assay. Bioactivity of ILRAP-IL-18 protein was demonstrated in the culture medium of transfected CHO cells. These data imply that the ILRAP-IL-18 construct shows potential for use in vivo, where cell secretion of protein is crucial.     

稳定表达犬细小病毒VP2结构蛋白的CHO-K1细胞系的建立

为构建犬细小病毒VP2基因分泌性表达细胞系,通过酶切将人CD5信号肽序列从质粒中切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法扩增犬细小病毒VP2基因,并将其插入到pcDNA3.1-CD5sp载体中CD5信号肽的下游,使其与CD5信号肽序列融合,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经脂质体介导转染细胞,后通过G418筛选,建立出稳定表达VP2蛋白的CHO-K1细胞系。测序结果表明,构建的犬细小病毒VP2基因的分泌型表达载体结构正确,表达载体经脂质体介导转染CHO-K1细胞,通过G418加压,筛选出稳定转染VP2基因的细胞株,经PCR检测证明VP2基因已经整合到细胞的染色体中;经RT-PCR、Westernblot分别检测VP2基因表达的mRNA和VP2蛋白,证明犬细小病毒VP2基因能够在CHO-K1细胞进行稳定性表达。这为下一步研究犬细小病毒VP2蛋白与宿主细胞的相互作用及VP2DNA疫苗奠定了基础。     

稳定表达犬瘟热病毒Nectin4受体的Vero细胞系构建

为提高病毒的分离效率,本试验设计构建了能够表达犬瘟热病毒（canine distemper virus,CDV）上皮细胞Nectin4受体的Vero细胞。为增加蛋白定位的准确性并易于鉴定,使用Igκ信号肽替换原有信号肽序列并添加了HA标签,在Nectin4 ORF后串联IRES-Puro序列并连入pCI-Neo真核表达载体,得到完整的转染载体pCI-N4。不同浓度嘌呤霉素孵育Vero细胞得到最小筛选浓度为6 μg/mL。pCI-N4重组质粒转染Vero细胞后使用6 μg/mL嘌呤霉素筛选,5~7 d后出现具有抗性的细胞簇,有限稀释法连续单克隆纯化3代后获得稳定表达的细胞系。构建细胞系传代至15代能检测到Nectin4 mRNA转录,Western blotting检测筛选细胞得到约60 ku目的蛋白表达,间接免疫荧光检测显示纯化细胞蛋白表达丰度高且表达均一,激光共聚焦观察Nectin4目的蛋白定位于细胞膜,说明筛选的Vero-Nectin4细胞系能够稳定表达,表达蛋白能够满足作为CDV受体的结构要求。临床CDV阳性病料经研磨滤菌处理后接种构建细胞系能够产生典型的合胞体细胞病变,Vero对照组盲传3次未有病变。分离毒株TICD₅₀=10^-5.9/0.1 mL。构建的Vero-Nectin4细胞系可用于CDV分离。     

Canine thyrotropin beta-subunit gene: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and transient expression in the Chinese hamster ovary cells

The gene encoding the mature β subunit of canine thyroid stimulating hormone (cTSHβ) was cloned, sequenced and expressed in Escherichia coli and in Chinese hamster ovary (CHO) cells, and monoclonal antibodies against the recombinant cTSHβ purified from E. coli were generated. The gene fragment that encodes mature TSHβ was cloned from the canine genomic DNA by direct polymerase chain reaction (PCR) using primers that were designed based on the consensus sequences from other species. The resulting 891 basepairs (bp) of genomic DNA consisted of two coding exons of the canine TSHβ gene and an intron of 450 bp. The two exons, which encode the mature cTSHβ subunit, was joined together by an overlap PCR and was expressed in E. coli as 6×His-tagged protein. The purified recombinant cTSHβ with a molecular weight of about 15 kDa was recognized by the polyclonal antibodies prepared against the native canine TSH in Western blot. Monoclonal antibodies were raised against the purified cTSHβ and subsequently characterized. For transient expression in CHO cells that are permanently transfected with the bovine common gene, a 60-oligonucleotide signal peptide coding sequence was added to the 5′ end of the cTSHβ gene before it was cloned into the mammalian expression vector pRSV and used to transfect CHO cells. The medium from these transfected cells, presumably containing the bovine and canine TSHβ in heterodimeric confirmation, exhibited TSH bioactivity as indicated by the stimulation of cAMP production in the cultured FRTL-5 thyrocytes.     

Expression,Identification and Subcellular Localization of V Protein of Canine Distemper Virus

In order to study the function of canine distemper virus (CDV) V protein,V gene fragment was inserted into the pGEX-6p-1 vector,and then pGEX-6p-1-CDV-V recombinant expression plasmid was obtained.Purified V protein immunized BALB/c mice to prepare the positive serum.Meanwhile,to confirm the distribution of V protein and subcellular localization with confocal laser technology,a eukaryotic expression recombinant plasmid pcDNA3.1-CDV-V was built,then transfected into Vero cells.The results showed that V protein was successfully expressed and the positive serum was prepared.The recombinant plasmid pcDNA3.1-CDV-V was expressed in Vero cells and mainly expressed in cytoplasm.The results laid a function for functional studies of CDV V protein.     

犬瘟热病毒V蛋白的表达、鉴定及亚细胞定位

为研究犬瘟热病毒（canine distemper virus,CDV） V蛋白的功能,将CDV V基因片段与pGEX-6P-1载体连接,构建pGEX-6P-1-CDV-V重组表达质粒,通过原核表达系统表达了V蛋白,并将纯化的V蛋白免疫BALB/c小鼠,制备阳性血清。同时,将CDV V基因片段与pcDNA3.1载体连接,构建pcDNA3.1-CDV-V重组表达质粒,经转染Vero细胞后,用激光共聚焦技术确定V蛋白在真核细胞中的分布及亚细胞定位。结果显示,成功表达了V蛋白,并制备了阳性血清。重组质粒pcDNA3.1-CDV-V在外源真核细胞Vero中获得了表达,表达蛋白主要聚集于细胞质。本试验结果为进行CDV V蛋白的功能研究奠定了基础。     

Cloning and Eukaryotic Expression Plasmid Construction of Porcine Interleukin-18 Gene

To obtain recombinant eukaryotic expression plasmid of porcine interleukin-18(IL-18), the whole gene of porcine IL-18 gene was amplified from porcine spleen, lung and lymph nodes by RT-PCR and cloned into eukaryotic expression vector pZJ-1. The recombinant expression plasmid pZJ-IL-18 were identified by enzyme digestion and sequencing analysis, and was transfected into 293T cells.The expression of IL-18 was detected by Real-time PCR and Western blotting in both gene and protein levels. The results showed that the eukaryotic expression plasmid of porcine IL-18 was constructed and could express transiently in 293T cells. Western blotting result confirmed that porcine IL-18 polyclonal antibody could react specifically with approximately 17 ku expression products,and indicated that IL-18 could express correctly and be responsive.This study constructed the eukaryotic expression plasmid of porcine IL-18 gene which could express transiently in 293T cells, and laid the foundation for studying function of IL-18.     

猪白细胞介素18基因的克隆及真核表达重组质粒的构建

为获得表达猪白细胞介素18（interleukin-18,IL-18）的真核表达重组质粒,试验通过RT-PCR从猪脾脏、肺脏和淋巴结组织中扩增猪IL-18全基因并定向克隆到真核表达载体pZJ-1,测序分析和酶切鉴定正确后,转染至293T细胞中,并通过实时荧光定量PCR和Western blotting分别在基因和蛋白水平检测IL-18的表达。结果表明,试验成功构建了真核表达重组质粒pZJ-IL-18,且可以在293T细胞中表达IL-18基因。Western blotting试验证实,猪IL-18多克隆抗体能与约17 ku的表达产物发生特异性反应,表明IL-18能正确表达且具有反应原性。本试验构建了表达猪IL-18基因的真核表达质粒,并在293T细胞中瞬时表达,为进一步研究IL-18的功能奠定基础。     

猪生长激素促分泌素受体基因真核表达载体的构建及鉴定简

本试验旨在构建猪生长激素促分泌素受体（pGHS-R）真核表达系统,并瞬时转染人源胚胎肾细胞（HEK293T）观察其表达情况。以猪基因组为模板,通过剪接重叠延伸聚合酶链式反应（SOE-PCR）克隆出pGHS-R的编码区序列,插入真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒pcDNA3.1(+)/pGHS-R,酶切鉴定并测序,加myc标签,瞬时转染HEK293T细胞,用Western blotting鉴定该重组质粒是否能在真核细胞中表达相应的目的蛋白。结果显示,本试验成功扩增出pGHS-R编码序列,酶切和测序结果表明pcDNA3.1(+)-myc/pGHS-R构建正确,Western blotting方法证实转染的该质粒能在HEK293T细胞中正确表达目的蛋白。结果表明,本试验成功构建了pGHS-R真核表达载体,并正确表达蛋白,为进一步研究GHS-R的功能奠定了基础。     

白细胞介素-2基因表达质粒对犬细小病毒VP2DNA疫苗在小鼠的免疫增强作用

犬细小病毒编码的VP2蛋白是该病毒主要抗原蛋白。研究证实由VP2基因制备的DNA疫苗能够刺激机体产生免疫应答反应。为提高VP2DNA疫苗的免疫原性,本研究在小鼠体内尝试了利用犬白细胞介素2（cIL-2）基因增强VP2DNA疫苗免疫应答的研究。通过RT-PCR方法从犬脾淋巴细胞中分别扩增含终止密码子和不合终止密码子的cIL-2cDNA基因,然后将基因插入到真核表达载体pcDNA3．1中,分别构建成非融合的和与Myc／His融合的clL-2基因真核分泌型表达载体,pcDNA-cIL-2和pcDNA-cIL-2／MH。将pcDNA-oIL-2／MH表达栽体通过磷酸钙方法转染HEK293T细胞进行瞬时表达,以确定构建的表达栽体能否介导cIL-2在真核细胞中进行分泌表达。然后用VP2表达载体（pcDNA-CD5sp-VP2,本室构建）单注射和VP2／IL-2表达载体共注射对小鼠进行免疫（用pcD-NA3．1栽体作为阴性对照）。免疫后通过ELISA方法检测免疫后不同时期小鼠血清VP2的抗体水平,并通过细胞增殖试验检测免疫后小鼠脾脏淋巴细胞的增殖反应,用ELISA方法测定小鼠淋巴细胞γ干扰素的表达水平。试验结果表明,扩增的小鼠cIL-2基因与GenBank的参考序列一致,构建的cIL-2表达载体能够介导重组cIL-2在HEK293T细胞中进行分泌表达。免疫结果显示,利用cIL-2／VP2表达载体共免疫小鼠,免疫后35d血清中VP2的抗体水平达到1：5120,明显高于VP2表达载体单免疫组（P〈0．01）。淋巴细胞增殖试验表明,2组免疫小鼠的淋巴细胞刺激指数均明显高于阴性对照组（P〈0．01）,共免疫组的刺激指数又明显高于单免疫组（P〈0．05）。共免疫小鼠淋巴细胞7干扰素的表达水平明显高于单免疫组和阴性对照组（P〈0．01）。由此可见,cIL-2表达载体可明显提高CPVVP2基因疫苗的免疫应答水平。     

Canine thyrotropin β-subunit gene: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and transient expression in the Chinese hamster ovary cells

The gene encoding the mature β subunit of canine thyroid stimulating hormone (cTSHβ) was cloned, sequenced and expressed in Escherichia coli and in Chinese hamster ovary (CHO) cells, and monoclonal antibodies against the recombinant cTSHβ purified from E. coli were generated. The gene fragment that encodes mature TSHβ was cloned from the canine genomic DNA by direct polymerase chain reaction (PCR) using primers that were designed based on the consensus sequences from other species. The resulting 891 basepairs (bp) of genomic DNA consisted of two coding exons of the canine TSHβ gene and an intron of 450 bp. The two exons, which encode the mature cTSHβ subunit, was joined together by an overlap PCR and was expressed in E. coli as 6×His-tagged protein. The purified recombinant cTSHβ with a molecular weight of about 15 kDa was recognized by the polyclonal antibodies prepared against the native canine TSH in Western blot. Monoclonal antibodies were raised against the purified cTSHβ and subsequently characterized. For transient expression in CHO cells that are permanently transfected with the bovine common α gene, a 60-oligonucleotide signal peptide coding sequence was added to the 5′ end of the cTSHβ gene before it was cloned into the mammalian expression vector pRSV and used to transfect CHO cells. The medium from these transfected cells, presumably containing the bovine α and canine TSHβ in heterodimeric confirmation, exhibited TSH bioactivity as indicated by the stimulation of cAMP production in the cultured FRTL-5 thyrocytes.     

犬TfR胞外区密码子的优化、真核表达及与犬细小病毒VP2蛋白结合

为制备大量具有天然活性的犬胞外区可溶性转铁蛋白受体(sTfR),本试验通过密码子优化提高sTfR在真核细胞中的表达水平.利用RT-PCR方法从犬肝脏中扩增sTfR编码基因,依据该基因编码的氨基酸序列,参照人偏爱的密码子,对该基因进行密码子优化并由公司合成.利用peDNA3.1-CD5质粒分别构建野生型和密码子优化的sTfR基因真核表达载体,经磷酸钙介导使其在HEK293T细胞中进行表达,利用Western-blotting鉴定表达产物,通过ELISA检测重组犬sTfR蛋白与犬细小病毒VP2蛋白的结合活性.结果显示本试验扩增的犬sTfR基因与GenBank该基因序列的同源性为100%;通过在HEK 293T细胞中进行瞬时表达,结果显示密码子优化可以明显提高sTfR基因在HEK 293T细胞中的表达水平,提高了75%.同时表达的sTfR蛋白能够与犬细小病毒VP2蛋白进行特异结合,表明表达的重组sTfR蛋白具有天然活性.     

牛新孢子虫NcSRS2基因腺病毒穿梭载体的构建及在293细胞中的表达

应用PCR技术扩增牛新孢子虫NcSRS2基因,纯化PCR产物后与克隆载体pMD18-T Simple Vector连接,将PCR、酶切鉴定及测序分析正确的pMD-18T-NcSRS2重组质粒进行EcoRⅠ和XbaⅠ双酶切,克隆至相同酶切回收后的腺病毒穿梭载体pCR259中,再将PCR、酶切鉴定正确的pCR259-NcSRS2重组质粒转染293细胞,应用IF-AT和Western-blotting技术检测重组质粒在293细胞中的表达情况。结果显示,扩增的牛新孢子虫NcSRS2基因长度为1 227bp,与GenBank中发表的NcSRS2（AF061249）核苷酸序列同源性为99%,构建的pCR259-NcSRS2重组质粒在293细胞中得到瞬时表达,表达蛋白的相对分子质量为43 000,具有较好的反应原性。本试验为新孢子虫病腺病毒载体疫苗的构建奠定了基础。     

山羊TLR2绿色荧光蛋白融合载体的构建

为了构建在哺乳动物细胞中表达的山羊TLR2绿色荧光蛋白融合载体,试验根据克隆到T载体上的山羊TLR2测序结果,设计1对不含终止子的引物,将山羊TLR2 PCR产物连接到pEGFP-N1载体上,用磷酸钙法转染293T细胞,并在荧光显微镜下观察。结果表明:重组质粒经酶切和测序鉴定正确,且在293T细胞中表达;融合蛋白发出绿色荧光,表明TLR2-EGFP主要分布在细胞膜上。说明试验成功地构建了pEGFP-TLR2-N1绿色荧光蛋白融合载体。     

猪白介素4在CHO-K1细胞中的重组表达及生物学活性分析

将猪白介素4(Porcine interleukin4,PIL-4)基因亚克隆到真核表达载体pEGFP-N1中,构建重组表达质粒pEGFP-PIL-4,利用脂质体法转染CHO-K1细胞,采用荧光显微镜实时观察、RT-PCR和Western-blot分别检测CHO细胞转录表达目的分子的情况,通过MTT法检测所表达蛋白的生物学活性。结果在将重组质粒转染CHO-K1细胞中24、48 h后的均观察到绿色荧光;经G418筛选14~20 d后转染的细胞形成了阳性细胞集落,用RT-PCR扩增出约339 bp的目的基因片段;经Western-blot检测到约为39 000的特异蛋白分子条带,并证明在转染重组质粒的细胞中表达了高生物活性的重组蛋白。     

犬白细胞介素18基因的克隆与序列分析

为研究犬白细胞介素18(IL-18)的生物学功能,从犬外周血中分离白细胞,经Con A刺激后,提取总RNA,通过反转录-聚合酶链反应(RT-PCR)扩增犬IL-18基因,连接pMD19-T simple vector,转化DH5α感受态细胞,经双酶切鉴定,获得阳性重组质粒后进行序列分析。结果表明,获得的犬IL-18基因全长为582bp,编码氨基酸194个。将犬与GenBank中牛、猫、羊、猪、鼠、兔、狐、貉IL-18基因进行同源性比较,发现犬IL-18与红狐和貉IL-18的同源性最高,氨基酸序列同源性分别达96.4%、91.2%,与其他物种则存在较大种属差异。     

基于甲病毒复制子的犬瘟热核酸疫苗的构建及表达性研究

本试验利用T-A克隆技术,构建克隆载体pEASY-Blunt-F,BamHⅠ酶切pEASY-Blunt-F,回收F基因,将其克隆至pSCA1真核表达载体中,获得重组质粒pSCA1-F。经DNA测序、限制性内切酶分析和PCR鉴定,结果表明重组质粒pSCA1-F成功构建。通过间接免疫荧光试验和Western blotting证实F基因在转染细胞中表达。此外,细胞凋亡的检测结果表明,pSCA1-F能引起转染的细胞发生凋亡。