Objective To assess the impact of freeze-drying and dry heat virus inactivation processes on the activity ofα2-macroglobulin(A2M)derived from human plasma Cohn fraction Ⅳ.Methods A2M derived from human plasma Cohn fraction Ⅳ was prepared and subjected to programmed freeze-drying with dry heat virus inactivation.The lyophilized products were evaluated for their appearance,water content,and validation of the viral inactivation process.The bioactivity of the products before and after lyophilization as well as before and after dry heat inactivation was determined via trypsin inhibition,and the comparisons were studied.Results The appearance of the lyophilized product was fluffy,and the water content was(5.83±0.45)％.The specific activities of the samples before and after lyophilization were(10.199±0.137)and(10.033±0.201)μg/mg,respectively,with no statistically significantdifference between the two groups(P＞0.05).The viral inactivation of the samples was carried out by using dry heat inactivation conditions at 100 ℃ for 30 min.After inactivation,the reduction was ≥5.125 LgTCID50/0.1 mL in Pseudorabies virus(PRV)titers,≥4.500 LgTCID50/0.1 mL in Sindbis virus(SinV)titers,≥6.375 LgTCID50/0.1 mL in encephalomyocarditis virus(EMCV)titers,and≥4.500 LgTCID50/0.1 mL in porcine parvovirus(PPV)titers.The specific activities of the samples before and after dry heat were(9.921±0.292)and(10.091±0.278)μ g/mg,respectively,with no statistically significant difference between the two groups.Conclusion A2M derived from human plasma Cohn fraction Ⅳ,when subjected to freeze-drying followed by dry heat inactivation at 100 ℃ for 30 minutes,can effectively inactivate viruses without altering the biological activity of the product.

Objective To construct one high-copy BAC vector with cloning capacity for large DNA fragment insertion and use it for the haplotype analysis of CYP2A6 gene.Methods Oligos including multiple cloning sites were annealed and ligated into the Hind Ⅲ/BamH Ⅰ site of pGEM-3Z and pBeloBACll,separately.Then,the intermediate vectors were digested by Hind Ⅲ and ligated together,so as to get the head-to-tail oriented high-copy BAC vector pBAC-BJH after the blue-white spotting test.STI PCR method was used for the amplification of whole CYP2A6 gene,and purified amplicon was double digested by BstB Ⅰ/Mlu Ⅰ and ligated into the same site of newly constructed vector pBAC-BJH.Several clones were then picked up and sequenced for the haplotype analysis of carriers with newly discovered CYP2A6 variant 355A＞T.Results One high-copy BAC vector pBAC-BJH with 7 newly added multiple cloning sites was successfully constructed.High copy number and multiple cloning sites were the advantages of this plasmid.With this vector,haplotype analysis result for 22C＞T,51A＞G,355A＞T in carrier with newly detected CYP2A6mutation was CGT.Conclusion One vector pBAC-BJH convenient for cloning large DNA fragment was successfully developed,and it can be used for the haplotype analysis of cytochrome p450 gene and cloning or sequencing of other genes with large genomic DNA inserts.

ObjectiveTo observe the effect of the Fangfeng Tongshengsan on post-chemoembolization syndrome with primary liver cancer or postoperative liver metastases of colorectal cancer. MethodSeventy-two patients suffered from post-chemoembolization syndrome after transcatheter hepatic arterial chemoembolization were randomly divided into 2 groups， including a Fangfeng Tongshengsan group and a control group， with 36 patients in each group. The patients in Fangfeng Tongshengsan group orally took the decoction for consecutive 7 d. The patients in the control group were physically cooled down with alcohol rub bath and ice pack for consecutive 7 d. Furthermore， the difference of fever， Karnofsky performance status （KPS）， pain in the liver region， nausea vomiting， constipation， and liver function between these two groups were observed. ResultCompared with the control group， Fangfeng Tongshengsan significantly relieved fever， reduced the body temperature （P<0.05）， and shortened the duration of fever （P<0.05）， indicating that Fangfeng Tongshengsan remarkably improved the KPS （P<0.05）. Meanwhile， Fangfeng Tongshengsan obviously alleviated nausea， vomiting， and constipation status and shortened the duration time compared with the control group （P<0.05）. In addition， the parameters of liver function including alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， γ-glutamyl transpeptidase （GGT）， and total bilirubin （TBIL） were significantly decreased in the Fangfeng Tongshengsan group （P<0.05）， which indicated that Fangfeng Tongshengsan alleviated liver dysfunction of patients with post-chemoembolization syndrome. ConclusionFangfeng Tongshengsan can be used to treat post-chemoembolization syndrome with primary liver cancer and postoperative liver metastases of colorectal cancer.