基于假病毒的高通量人类免疫缺陷病毒表型耐药性检测方法的建立和优化

Establishment and optimization of a high throughput phenotypic test for the detection of drug resist-ance in human immunodeficiency virus（HIV）strains

目的：建立一种高通量人类免疫缺陷病毒表型耐药性检测方法。方法通过酶切连接方式失活荧光素酶基因，用LacZ基因替代原有的蛋白酶和逆转录酶基因。通过PCR方法从含耐药基因的pSG3△env质粒中扩增pol基因，用infusion定向克隆技术连接入酶切后的pNL4-3.Lac，对该表型耐药性检测方法的主要影响因素进行优化。结果构建了用于高通量耐药性检测的pNL4-3.Lac，确定了检测方法中细胞加入量为10000细胞/孔（96孔板），病毒加入量为200 TCID 50/孔，DEAE-dextran的最佳工作浓度为15μg/ml。用12种抗病毒药物检测两种假病毒8次，确定方法具有良好的重复性（CV值在4.32％～28.46％之间）。构建了6种不同的假病毒与基于pSG3△env的耐药性检测方法进行比较，两者之间具有良好的一致性。结论基于pNL4-3.Lac的表型耐药性检测方法结合了pSG3△env和pNL4-3检测系统的优点，能高通量评价HIV病毒对药物的耐受性，可用于感染者耐药性分析和抗病毒药物的筛选。

Objective To establish a high throughput phenotypic test for the detection of drug resistance in human immunodeficiency virus（HIV）strains. Methods The gene encoding luciferase was inactivated through restriction enzyme digestion and ligation. LacZ gene was used to replace the genes encoding original protease and reverse transcriptase. pol genes were amplified from pSG3△env plasmid and cloned into a new backbone plasmid through infusion. The factors that might affect the results of the test were optimized. Results The parental backbone plasmid pNL4-3. Lac was constructed,of which the gene encoding luciferase was inactivated and bearing the LacZ gene instead of genes encoding protease and reverse transcriptase. Several influential factors including cell numbers（10 000 / well）,virus inoculation（200 TCID50 /well）and the concentration of DEAE-dextran（15 μg/ ml）were optimized. The reproducibility of this test was confirmed by testing 12 anti-HIV drugs against 2 pseudovirus strains 8 times,presenting the coefficient of variations（CVs）from 4. 32% to 28. 46% . Six types of pseudovirus were constructed and tested against the 12 anti-HIV drugs,the results of which were compared with those by using the pSG3△env-based pseudovirus test. The results of the two tests presented good consistency. Conclusion The high throughput phenotypic test based on pNL4-3. Lac plasmid,combining the advantages of pSG3△env and pNL4-3 systems, could be used to analyze the drug resistance patterns of HIV-1 infectors and screen new drugs for antiretroviral therapy in a rapid and effective way.