變異乙型肝炎表面抗原在畢赤酵母中的克隆表達(dá)及表面抗原診斷試劑的評(píng)價(jià)
發(fā)布時(shí)間：2018-07-21 12:48
【摘要】： 乙型肝炎是一種對(duì)公共健康危害嚴(yán)重的疾病。近年來隨著乙型肝炎疫苗和抗病毒藥物的廣泛使用，乙型肝炎病毒的感染和傳播得到了有效的防止。但是，乙型肝炎的防治是一項(xiàng)長期而艱巨的任務(wù)，隨著對(duì)乙肝病毒研究的深入，一些新的問題不斷被發(fā)現(xiàn)，，如免疫后人群，抗病毒藥物治療的患者中以及慢性HBV感染的人群中都出現(xiàn)了HBV表面抗原的變異。這些變異對(duì)免疫診斷試劑提出了新的挑戰(zhàn)。大量文獻(xiàn)報(bào)道了診斷試劑對(duì)變異表面抗原的漏檢。但是國內(nèi)對(duì)HBV變異的流行病學(xué)及變異對(duì)國產(chǎn)診斷試劑影響的研究還較少。 本研究第一部分利用已構(gòu)建好的野生型及3個(gè)HBV S區(qū)變異的質(zhì)粒(T126N，D144A，G145R)作為模板，設(shè)計(jì)含有特定酶切位點(diǎn)的引物，進(jìn)行PCR后用EcoRⅠ和NotⅠ雙酶切回收的PCR產(chǎn)物，連接入酵母表達(dá)質(zhì)粒pPICZA和pPICZα，構(gòu)建出HBV S區(qū)野生及變異酵母表達(dá)質(zhì)粒。測序結(jié)果顯示，讀碼框架及核苷酸序列均正確。 利用SalⅠ酶切構(gòu)建好的6種表達(dá)質(zhì)粒(胞內(nèi)表達(dá)4種，分泌表達(dá)2種)使之成為線性片斷。電穿孔轉(zhuǎn)化P．pastroris酵母細(xì)胞GS115，用高濃度抗生素Zeocine(2000ng／ml)加壓篩選出高效表達(dá)的含有多拷貝整合的菌株。 對(duì)于篩選出的高抗性的菌株，經(jīng)甲醇誘導(dǎo)表達(dá)后，用玻璃珠破碎法裂解酵母細(xì)胞，釋放菌體內(nèi)的表達(dá)產(chǎn)物。各菌株的裂解液稀釋相同的倍數(shù)，用雅培公司的HBsAg試劑ARCHETECT對(duì)表達(dá)的HBsAg定量，并利用確證試劑進(jìn)行確證，結(jié)果均為陽性。選擇表達(dá)量高的菌株進(jìn)行大量表達(dá)。表達(dá)產(chǎn)物以多克隆抗體作為一抗，使用免疫印跡法(Western blot，WB)檢測4種表達(dá)產(chǎn)物，結(jié)果證明表達(dá)產(chǎn)物為HBsAg。 本研究的第二部分是利用各種HBsAg對(duì)國內(nèi)外診斷試劑進(jìn)行評(píng)價(jià)。為了確保實(shí)驗(yàn)結(jié)果的可靠性，排除由于實(shí)驗(yàn)操作問題對(duì)結(jié)果的影響，我們對(duì)ELISA實(shí)驗(yàn)操作進(jìn)行了規(guī)范，選擇了一份HBV表面抗原強(qiáng)陽性血清倍比稀釋后，作為檢測用血樣。利用各家診斷試劑對(duì)血樣于不同時(shí)間進(jìn)行了10次檢測，最后結(jié)果統(tǒng)計(jì)顯示試驗(yàn)的重復(fù)性及精確性都己達(dá)到要求。并且，在以后利用變異抗原對(duì)診斷試劑進(jìn)行評(píng)價(jià)時(shí)，仍然將此血清作為內(nèi)標(biāo)以控制試驗(yàn)操作的一致性。 將酵母表達(dá)的抗原和我們收集到的CHO細(xì)胞和小鼠L細(xì)胞表達(dá)的抗原稀釋到一定的濃度，用各種HBsAg診斷試劑進(jìn)行檢測，對(duì)國內(nèi)外診斷試劑進(jìn)行評(píng)價(jià)。結(jié)果發(fā)現(xiàn)國產(chǎn)診斷試劑對(duì)HBV野生型不同亞型的檢測靈敏度與進(jìn)口試劑相差了約4-32倍。而對(duì)多種不同變異抗原的檢測，4家國產(chǎn)試劑均存在一定的漏檢情況。并且，利用不同表達(dá)系統(tǒng)表達(dá)的變異抗原對(duì)診斷試劑的評(píng)價(jià)結(jié)果也是基本相同的。國產(chǎn)試劑對(duì)G145R變異及其相關(guān)的多點(diǎn)變異的檢測能力弱；對(duì)T118K／P120Q，Q129R／M133T等聯(lián)合變異的檢測能力亦有待于提高；而對(duì)于T126N，D144A等單點(diǎn)變異國內(nèi)外試劑的檢測能力基本一致。同時(shí)，用含有1／4 G145R變異的血樣對(duì)診斷試劑進(jìn)行評(píng)價(jià)，發(fā)現(xiàn)與以上相同的結(jié)論，國產(chǎn)試劑的檢測靈敏度下降。
[Abstract]:Hepatitis B is a serious disease to public health. With the widespread use of hepatitis B vaccine and antiviral drugs in recent years, the infection and transmission of hepatitis B virus have been effectively prevented. However, the prevention and control of hepatitis B is a long-term and arduous task. With the in-depth study of HBV, some new types of hepatitis B virus have been studied. Problems have been discovered, such as the immunization of the population, in the patients with antiviral drugs and of the HBV surface antigen variation in the people with chronic HBV infection. These variations pose a new challenge to the immunodiagnostic reagents. A large number of documents have reported the leakage of the diagnostic reagents to the variant surface antigenic agents. However, the epidemic of HBV variation in China has been reported. There are few studies on the effects of learning and mutation on domestic diagnostic reagents.
In the first part of this study, using the constructed wild type and 3 HBV S region variant plasmids (T126N, D144A, G145R) as a template, the primers containing specific enzyme cut sites were designed and the PCR products recovered by EcoR I and Not I were reclaimed after PCR. The yeast expressed plasmid pPICZA and pPICZ alpha were connected to the yeast expression plasmid pPICZA and pPICZ alpha, and a wild and variant yeast table was constructed. Plasmid sequencing and sequencing showed that the coding frame and nucleotide sequence were correct.
6 kinds of expression plasmids (4 kinds of intracellular expression and 2 kinds of secretory expression) formed by Sal I enzyme were used as linear fragments. Electroporation was used to convert P.pastroris yeast cell GS115, and high concentration antibiotic Zeocine (2000ng / ml) was used to screen the highly expressed strains with multi copy integration.
After the screening of highly resistant strains, the yeast cells were cleaved by glass bead breakup and the expression products were released by glass bead breakage. The lysate of each strain was diluted the same multiplier. The expression of HBsAg was quantified by the HBsAg reagent ARCHETECT of the Abbott Company and confirmed by the reagents. The results were all positive. Western blot (WB) was used to detect 4 kinds of expression products, and the result showed that the expression product was HBsAg..
The second part of this study is to evaluate the domestic and foreign diagnostic reagents using various HBsAg. In order to ensure the reliability of the experimental results and eliminate the effect of the experimental operation on the results, we have standardized the experimental operation of ELISA, and selected a HBV surface antigen strong positive serum double dilution, as a detection blood sample. 10 tests were carried out on the blood samples at different times. The results of the results showed that the repeatability and accuracy of the test had reached the requirement. And, when the diagnostic reagent was evaluated by using the variant antigen, the serum was still used as the internal standard to control the consistency of the experiment.
The antigen expressed by yeast and the antigen expressed by the CHO and L cells of the mice were diluted to a certain concentration, and the diagnostic reagents were evaluated by various HBsAg diagnostic reagents. The results showed that the detection sensitivity of the domestic diagnostic reagents to the different subtypes of HBV wild type was about 4-32 times that of the imported reagents. The 4 homemade reagents were detected by 4 homemade reagents, and the results of the diagnostic reagents expressed by different expression systems were basically the same. The ability of the domestic reagents to detect the variation of G145R and its related multipoint mutations was weak; T118K / P120Q, Q129R / M133T, etc. The detection ability of the mutation is still to be improved, and the detection ability of the domestic and foreign reagents of T126N, D144A and other single variation is basically the same. At the same time, the diagnostic reagents are evaluated with the blood samples containing 1 / 4 G145R variation, and the same conclusion is found.
【學(xué)位授予單位】：中國藥品生物制品檢定所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392
[Abstract]:Hepatitis B is a serious disease to public health. With the widespread use of hepatitis B vaccine and antiviral drugs in recent years, the infection and transmission of hepatitis B virus have been effectively prevented. However, the prevention and control of hepatitis B is a long-term and arduous task. With the in-depth study of HBV, some new types of hepatitis B virus have been studied. Problems have been discovered, such as the immunization of the population, in the patients with antiviral drugs and of the HBV surface antigen variation in the people with chronic HBV infection. These variations pose a new challenge to the immunodiagnostic reagents. A large number of documents have reported the leakage of the diagnostic reagents to the variant surface antigenic agents. However, the epidemic of HBV variation in China has been reported. There are few studies on the effects of learning and mutation on domestic diagnostic reagents.
In the first part of this study, using the constructed wild type and 3 HBV S region variant plasmids (T126N, D144A, G145R) as a template, the primers containing specific enzyme cut sites were designed and the PCR products recovered by EcoR I and Not I were reclaimed after PCR. The yeast expressed plasmid pPICZA and pPICZ alpha were connected to the yeast expression plasmid pPICZA and pPICZ alpha, and a wild and variant yeast table was constructed. Plasmid sequencing and sequencing showed that the coding frame and nucleotide sequence were correct.
6 kinds of expression plasmids (4 kinds of intracellular expression and 2 kinds of secretory expression) formed by Sal I enzyme were used as linear fragments. Electroporation was used to convert P.pastroris yeast cell GS115, and high concentration antibiotic Zeocine (2000ng / ml) was used to screen the highly expressed strains with multi copy integration.
After the screening of highly resistant strains, the yeast cells were cleaved by glass bead breakup and the expression products were released by glass bead breakage. The lysate of each strain was diluted the same multiplier. The expression of HBsAg was quantified by the HBsAg reagent ARCHETECT of the Abbott Company and confirmed by the reagents. The results were all positive. Western blot (WB) was used to detect 4 kinds of expression products, and the result showed that the expression product was HBsAg..
The second part of this study is to evaluate the domestic and foreign diagnostic reagents using various HBsAg. In order to ensure the reliability of the experimental results and eliminate the effect of the experimental operation on the results, we have standardized the experimental operation of ELISA, and selected a HBV surface antigen strong positive serum double dilution, as a detection blood sample. 10 tests were carried out on the blood samples at different times. The results of the results showed that the repeatability and accuracy of the test had reached the requirement. And, when the diagnostic reagent was evaluated by using the variant antigen, the serum was still used as the internal standard to control the consistency of the experiment.
The antigen expressed by yeast and the antigen expressed by the CHO and L cells of the mice were diluted to a certain concentration, and the diagnostic reagents were evaluated by various HBsAg diagnostic reagents. The results showed that the detection sensitivity of the domestic diagnostic reagents to the different subtypes of HBV wild type was about 4-32 times that of the imported reagents. The 4 homemade reagents were detected by 4 homemade reagents, and the results of the diagnostic reagents expressed by different expression systems were basically the same. The ability of the domestic reagents to detect the variation of G145R and its related multipoint mutations was weak; T118K / P120Q, Q129R / M133T, etc. The detection ability of the mutation is still to be improved, and the detection ability of the domestic and foreign reagents of T126N, D144A and other single variation is basically the same. At the same time, the diagnostic reagents are evaluated with the blood samples containing 1 / 4 G145R variation, and the same conclusion is found.
【學(xué)位授予單位】：中國藥品生物制品檢定所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前9條
1 趙翔,霍克克,李育陽;畢赤酵母的密碼子用法分析[J];生物工程學(xué)報(bào);2000年03期
2 劉忠淵,張富春,毛新芳,王蕓;利用畢赤酵母表達(dá)外源蛋白的研究[J];生物技術(shù);2004年01期
3 李洪釗,李亮助,孫強(qiáng)明,Q曖
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